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DIAGNOSTIC METHODS 



WEBSTER 



DIAGNOSTIC METHODS 

CHEMICAL, BACTERIOLOGICAL 
AND MICROSCOPICAL 



A Text-book for Students and Practitioners 



BY 

RALPH W. WEBSTER, M. D., Ph. D. 

ASSISTANT PROFESSOR OF PHARMACOLOGICAL THERAPEUTICS AND INSTRUCTOR IN MEDICINE IN 

RUSH MEDICAL COLLEGE, UNIVERSITY OF CHICAGO; DIRECTOR OF CHICAGO 

LABORATORY, CLINICAL AND ANALYTICAL 



FIFTH EDITION, REVISED AND ENLARGED 

WITH 37 COLORED PLATES 

AND 171 OTHER ILLUSTRATIONS 



PHILADELPHIA 

P. BLAKISTON'S SON & CO. 

1012 WALNUT STREET 



n/fc 



First Edition, Copyright, 1909, By P. Blakiston's Son & Co. 
Second Edition, Copyright, 191 2, By P. Blakiston's Son & Co. 
Third Edition, Copyright, 1913, By P. Blakiston's Son & Co. 
Fourth Edition, Copyright, 1914, By P. Blakiston's Son & Co. 
Fifth Edition, Copyright, 1916, By P. Blakiston's Son & Co. 




THE. MAPLE. PRESS- YORK- PA 



3CI.A427320 



*r 



TO THE MEMORY 

OF 

MY FATHER 

DR. JOHN RANDOLPH WEBSTER 

THIS VOLUME IS 
LOVINGLY DEDICATED 



PREFACE TO THE FOURTH EDITION 



Although, less than a year has elapsed since the appearance of the third edi- 
tion of this work, the author has felt justified in adding considerable new material 
in this revision. This has come about largely through the suggestions of teachers 
and general practitioners regarding the inclusion of certain subjects not hitherto 
discussed. 

Thus, the new matter embraces, among other subjects, a discussion of the 
Negri bodies recently successfully cultivated by Noguchi; Weisz's test for 
urochromogen; Dorrance's method for coagulation of the blood; Kowarsky's 
method for glucose in the blood; Levaditi and Manouelian's as well as Noguchi' s 
methods for staining spirochete in tissue; Lange's colloidal-gold test for con- 
genital syphilis; a discussion of the organism of anterior poliomyelitis recently 
cultivated by Flexner and Noguchi. The Wassermann test has been brought 
up to date by inclusion of considerable new matter; while the complement- 
fixation test in gonorrhea has been elaborated. The Herman-Perutz reaction 
for syphilis is fully described. The application of Abderhalden's dialyzation 
method to the diagnosis of cancer and dementia precox has been freely discussed. 
Further, an entirely new chapter on Clinical Bacteriology has been added, in 
which are discussed the differentiation of the more important pathogenic organ- 
isms and, also, the methods of preparation of vaccines. The subject matter 
throughout the text has been brought up to date by the inclusion of a large 
number of references to the original literature. 

The author wishes to express his great satisfaction at the reception of the third 
edition of his work and, also, his full appreciation of the kind suggestions offered 
him regarding the general matter of the book. He wishes, especially, to thank 
his colleagues Drs. T. L. Dagg and C. C. Croy for their many valuable sug- 
gestions and criticism. 

Ralph W. Webster. 
25 E. Washington St., Chicago. 



IX 



PREFACE TO THE FIRST EDITION 



In the present work the writer has attempted to bring together, for the 
use of the student and practitioner, the generally accepted facts regarding the 
various phases of clinical medicine, which may be rather more closely studied 
by the application of laboratory methods than without their use. It is to be 
especially emphasized that laboratory work must go hand in hand with the 
more direct clinical examination of the patient, as the former can be inter- 
preted only in the light of the latter. While it is true that, in some cases, the 
laboratory findings may be of even more value than those of physical examina- 
tion, yet it is to be understood that the function of the Clinical Laboratory is, 
more largely, perhaps, an accessory one, the application of methods of physical 
diagnosis usually pointing out the way toward a successful solution of the clinical 
problem by special laboratory methods, which yield numerous confirmatory 
or differential points not at all clearly defined by the methods of direct clinical 
examination. 

The aim of the author has been to present the direct bearing of the various 
methods outlined upon the clinical history of the case and to point out the 
special interpretation of the findings in any given examination. Particular 
attention has been directed to the selection of methods, both for the simpler 
clinical and more complex scientific requirements. As no method, no matter 
how exact it may be or how sound its basic principle, will yield reliable re- 
sults in the hands of the inexperienced, the writer has paid much attention to 
the details of such methods and has endeavored to direct the thought of the worker 
to the possible obstacles to be overcome before he is able properly to perform 
these examinations and interpret his results. 

The writer makes no claim for originality except, possibly, in the matter of 
arrangement of subject-matter and selection of methods and ideas, which have 
been established by others after years of earnest research. His endeavor has 
been to be as catholic as possible in his reading and as selective as his judgment 
permitted, to the end that the student or practitioner be saved the burden of 
sifting the wheat from the chaff. 

Numerous text-books, monographs, and special articles have been freely 
used in the preparation of the text, the writer attempting in each case to give 
due credit for such reference. It is possible that some direct use of material 
has been made which has not received deserved recognition. If so, the writer 
here acknowledges his indebtedness to them as well as to those whom he has 
directly quoted. 

It has seemed desirable to omit extensive reference to the literature, as a bib- 
liography to be of working value must be much more extensive and complete 



Xll PREFACE. 

than is possible within the scope of this book. The writer has, however, in- 
serted at the end of each chapter a list of the more important larger works 
which he has found useful in correlating the general subjects included in the 
several sections. 

In conclusion, the writer wishes to express his deep obligation to his col- 
leagues for their many valuable suggestions as to the subject-matter of the text: 
to Prof. W. S. Haines for assistance in revising the section on Urine; to Dr. 
J. M. Washburn for many additions and revisions of the section on Blood; to 
Dr. O. J. West for many practical points throughout the whole work; to Dr. 
W. A. Pusey for photographs of Blastomycetes under various conditions; to 
Dr. Brown Pusey for slides showing various organisms in the conjunctival 
exudates; and to Dr. N. Gildersleeve, of the University of Pennsylvania, for il- 
lustrations of megalosporon and microsporon. Further, the writer wishes to 
express his thanks to Miss Hill for the excellent work done by her in preparing 
the original drawings which appear throughout the work. 

Ralph W. Webster. 



TABLE OF CONTENTS. 



CHAPTER I. 
The Sputum. 

Page 

I. General Considerations i 

II. Physical and Chemical Characteristics 2 

Amount 2 

Consistency 2 

Reaction 3 

Color 3 

Odor 4 

Character 4 

Chemistry 5 

III. Macroscopic Examination 6 

Cheesy masses 6 

Dittrich's plugs 6 

Curschmann's spirals 7 

Fibrinous casts 8 

Concretions 8 

Bronchiolitis 8 

Pneumoliths 8 

Echinococcus membranes 9 

Foreign bodies 9 

IV. Microscopic Examination . 9 

Pus-cells 10 

Red blood-cells 11 

Epithelial cells . . 11 

Elastic tissue 12 

Crystals . . 13 

Bacteria 14 

Saprophytes 14 

Pathogenic types 17 

Tubercle bacillus 17 

Lepra bacillus 24 

Smegma bacillus 24 

Timothy bacillus 24 

Pneumococcus 25 

Friedlander's bacillus 25 

Influenza bacillus 26 

Bacillus pertussis 26 

Bacillus typhosus 26 

Staphylococcus and streptococcus pyogenes .... 26 

Bacillus pestis 27 

Bacillus anthracis 27 

Bacillus mallei 27 

Actinomyces hominis 27 

xiii 



Xiv TABLE OF CONTENTS. 

Pagb 

Animal parasites 28 

Amebae 28 

Flagellates 28 

Cestodes 29 

Trematodes 29 

V. The Sputa in Disease 30 

Pulmonary tuberculosis 30 

Croupous pneumonia 3 1 

Broncho-pneumonia 31 

Acute bronchitis 31 

Chronic bronchitis 32 

Simple type 32 

Putrid type 32 

Fibrinous type 32 

Bronchial asthma 33 

Influenza 33 

Gangrene of the lung 33 

Abscess of the lung 3 s 

Perforating empyema 34 

Pneumonoconioses . 34 



CHAPTER II. 

Oral, Nasal, Aural and Conjunctival Secretions. 

I. Oral Secretion 35 

General considerations 35 

Microscopic examination 36 

Pathologic changes 37 

Pharyngomycosis leptothrica 38 

Diphtheria 38 

Vincent's angina 4° 

Streptococcic sore throat 40 

Gonorrheal stomatitis 4 1 

Thrush 41 

Oral endamebiasis 42 

II. Nasal Secretion 44 

General considerations 44 

Pathologic changes 44 

Rhinitis 45 

Hay fever 45 

Meningitis 45 

III. Aural Secretion 46 

General considerations 46 

Pathologic changes 46 

IV. Conjunctival Secretions 47 

General considerations 47 

Pathologic changes 47 

Diphtheritic conjunctivitis 47 

Infectious conjunctivitis 47 

Gonorrheal conjunctivitis 48 

Trachoma 48 

Vernal conjunctivitis 49 



TABLE OF CONTENTS. XV 

CHAPTER III. 
Gastric Contents. 

Page 

I. General Considerations 50 

II. Methods of Obtaining Gastric Contents 52 

Stomach-tube 52 

Test meals 54 

Ewald meal 54 

Boas meal 55 

Riegel meal 55 

Fischer meal 55 

Salzer meal 56 

Sahli meal 56 

III. Macroscopic Examination 57 

Amount 57 

Color 58 

Odor 59 

Consistency 59 

Contents from fasting stomach 60 

Vomitus 60 

Contents after test meals 61 

IV. Microscopic Examination 62 

General 62 

Food remnants 62 

Boas-Oppler bacillus 62 

Sarcinae ventriculi 63 

Protozoa 63 

Tissue fragments 63 

Crystals 63 

V. Chemical Examination 63 

General 64 

Total acidity 64 

Free hydrochloric acid 65 

Qualitative tests 66 

Topfer's test 66 

Giinzburg's test 66 

Boas' test 67 

Tropeolin test 67 

Quantitative tests 67 

Mintz's method 68 

Topfer's method ' 68 

Amount of free hydrochloric acid 69 

Euchlorhydria 70 

Hypochlorhydria 70 

Ana-chlorhydria 70 

Hyperchlorhydria 71 

Combined hydrochloric acid 71 

Method of Martius and Luttke . 71 

Method of Topfer 7 2 

Hydrochloric acid deficit 73 

Organic acids 73 

Total organic acids 73 

Lactic acid 74 

UfTelmann's test 75 



Xvi TABLE OF CONTENTS. 

Pagb 

Kelling's test 75 

Strauss' method 75 

Butyric acid 76 

Acetic acid . 77 

Gastric ferments 77 

Pepsin 77 

Qualitative methods 78 

Quantitative examination 79 

Hammerschlag's method 79 

Mett's method 79 

Method of Thomas and Weber 80 

Chymosin 81 

Leo's method 81 

Riegel's method 81 

Lipase 82 

Products of protein digestion 82 

Products of carbohydrate digestion 82 

Blood 83 

Gases 83 

Function of the stomach and contents 84 

VI. Motility op the Stomach 84 

Leube's method 85 

Boas' method 85 

Method of Ewald and Sievers 85 

Winternitz' test 86 

VII. Absorptive Power of the Stomach 86 

Potassium iodide test 86 

VIII. Indirect Examination of the Stomach Contents 87 

Gunzburg's method 87 

Sahli's desmoid reaction 87 

IX. Gastric Juice in Disease 88 

Hyperchlorhydria 88 

Hypersecretion 89 

Achylia gastrica 89 

Acute gastritis 90 

Chronic gastritis 90 

Nervous dyspepsia 90 

Ulcer of the stomach 91 

Carcinoma of the stomach 91 

Salomon's test 93 

Neubauer and Fischer's test 93 

Wolff and Junghans' test 95 



CHAPTER IV. 

The Feces. 

I. General Considerations 99 

Normal feces 99 

Diet of Schmidt and Strasburger 100 

Diet of Folin 101 

Obtaining intestinal juice 101 

Functions of the intestinal juice 102 

Estimation of intestinal digestion 103 



TABLE OF CONTENTS. XV11 

Page 

II. Macroscopic Examination 103 

Method 103 

Amount . 104 

Consistency and form 105 

Odor 106 

Color 106 

Blood 108 

Mucus in 

Pus 113 

Food remnants 113 

Protein residues 114 

Fat residues .' 115 

Carbohydrate residues. 116 

Biliary constituents 117 

Intestinal sand and concretions 118 

Tissue fragments 118 

III. Microscopic Examination 119 

Technic 119 

Morphological elements 119 

Crystals 120 

IV. Chemical Examination 121 

Reaction 121 

Total solids 121 

Total nitrogen 122 

Fat 123 

Carbohydrates 123 

Phenoltetrachlorphthalein test 125 

V. Bacteriology of the Feces 128 

Technic 128 

Cholera spirillum 129 

Typhoid bacillus 130 

Method of Drigalski and Conradi ...130 

Method of Kendall and Day . 131 

Bacillus of Dysentery 132 

Tubercle bacillus 132 

VI. Parasitology of the Feces . . . 133 

Technic 133 

Protozoa 134 

Rhizopoda 134 

Amebina , 134 

Ameba coli 134 

Entameba coli 137 

Sporozoa 137 

Coccidium hominis . 137 

Flagellata 137 

Trichomonas intestinalis 137 

Cercomonas hominis 138 

Megastoma entericum 138 

Infusoria 138 

Balantidium coli . 138 

Entozoa 139 

Platodes. . . 139 

Cestodes. 139 

Taeniidae. . 140 



XV111 TABLE OF CONTENTS. 

Pagb 

Taenia solium 140 

Taenia saginata 141 

Taenia cucumerina 141 

Taenia nana 142 

Taenia diminuta 142 

Taenia echinococcus 143 

Bothriocephaloidea 144 

Bothriocephalus latus 144 

Dibothriocephalus cordatus 144 

Bothriocephalus sp. Ijima et Kurimoto . . 145 

Trematodes 145 

Nematodes 145 

Ascaridae 145 

Ascaris lumbricoides 145 

Ascaris mystax 146 

Oxyuris vermicularis 147 

Angiostomidae 147 

Strongyloides intestinalis 147 

Trichotrachelidae 149 

Trichiuris trichiura 149 

Trichinella spiralis 149 

Strongylidae 151 

Uncinaria duodenalis 151 

Uncinaria Americana 152 

Pseudo-parasites 153 



CHAPTER V. 

Parasites. 

I. General Considerations 156 

II. Trematodes 156 

Fasciolidae 157 

Fasciola hepatica 157 

Fasciolopsis Buski 158 

Opisthorchis felineus 159 

Opisthorchis sinensis 159 

III. Nematodes 160 

Eustrongylus gigas 160 

IV. Parasites op the Skin 161 

Arthropoda 161 

Arachnoidea 162 

Sarcoptes scabiei 162 

Demodex folliculorum 162 

Leptus autumnalis 162 

Insecta 163 

Hemiptera 163 

Pediculus capitis 163 

Pedi cuius vestimenti - 163 

Pediculus pubis 163 

Cimex lectularius 164 

Diptera 165 

Pulex irritans 165 

Pulex penetrans 165 



TABLE OF CONTENTS. XIX 

Pagb 

Vegetable Parasites 165 

Achorion Schonleinii 165 

Trichophyton megalosporon endothrix 166 

Microsporon Audouini 167 

Microsporon furfur 167 

Microsporon minutissimum 169 

Blastomycetes 169 

Sporothrix Schenckii 171 

Negri Bodies 172 

CHAPTER VI. 
The Urine. 

I. General Considerations 175 

Collection and preservation of the urine 176 

II. Physical Properties 177 

Quantity 177 

Polyuria 178 

Oliguria 179 

Anuria 180 

Appearance 180 

Color 181 

Odor 183 

Reaction 184 

Folin's method for total acidity 185 

Free mineral and organic acidity 185. 

Specific gravity 187 

Technic 188 

Rough estimate of total solids 189 

Optical activity 190 

III. Chemical Properties 190 

Normal composition 190 

Total solids and total ash . . 192 

Inorganic constituents . " 192 

Chlorids . 192 

Estimation of the chlorids 195 

Quantitative determination 195. 

Volhard's method 195 

Purdy's centrifugal method 198 

Phosphates 198 

Estimation of phosphates 202 

Quantitative determination 202 

Uranium method 202 

Total phosphoric acid . 205 

Purdy's centrifugal method 205 

Sulphur compounds 206 

Preformed sulphates 206 

Ethereal sulphates 207 

Neutral sulphur 208 

Estimation of total sulphur 209 

Folin's method . 209 

Determination of total sulphates 210 

Folin's method 21a 



XX TABLE OF CONTENTS. 



Page 

Determination of inorganic sulphates 210 

Purdy's centrifugal method 210 

Carbonates 211 

Sodium and potassium 211 

Calcium and magnesium 211 

Iron 213 

Organic constituents 213 

Nitrogenous bodies 213 

Total nitrogen 213 

Kjeldahl's method 217 

Urea 219 

Determination of urea 221 

Knop-Hufner method 222 

Doremus ureometer 222 

Folin's method 223 

Morner-Sjoqvist method 225 

Urease method .225 

Ammonia 227 

Quantitative determination 228 

Method of Schlosing 229 

Folin's method 230 

Formalin method 231 

Uric acid 232 

Quantitative determination 235 

Folin's method 235 

Salkowski-Ludwig method 236 

Method of Rudisch and Kleeberg . . . 237 

Ruhemann's method 239 

Purin bases 240 

Creatinin 241 

Qualitative tests 243 

Weyl's test 243 

Jaffa's test 243 

Quantitative determination 243 

Folin's methods 243 

Undetermined nitrogen 246 

Amino acids 246 

Hippuric acid 248 

Oxyproteic and alloxyproteic acids .... 248 

Allantoin 248 

Fatty acids 249 

Oxalic acid 250 

Quantitative determination 251 

Baldwin's method 251 

Ferments 251 

Pepsin 251 

Diastase 251 

Lipase 252 

Mucin-like bodies 252 

Mucin. 252 

Nucleo-albumin 252 

Pigments and chromogens 254 

Urochrome 254 

Uroerythrin 254 



TABLE OF CONTENTS. xxi 

Paob 

Urobilin 255 

Indican 256 

Tests for indican 257 

Jaffe's test 257 

Obermayer's test 257 

Rosenbach's test 258 

Quantitative determination 259 

Wang's method 259 

Folin's method 260 

Uroroseinogen 260 

Abnormal Composition 260 

Proteins 260 

Serum-albumin 261 

Albuminuria 261 

Functional 261 

Febrile 264 

Traumatic 265 

Hematogenous 265 

Toxic 265 

Neurotic 265 

With definite renal lesions 265 

Qualitative tests 266 

Heat and acid test 267 

Heller's nitric acid test 268 

Ferrocyanide test 271 

Sulpho-salicylic acid test 271 

Spiegler's test 271 

Quantitative methods 272 

Scherer's method 272 

Esbach's method 272 

Method of Tsuchiya 273 

Purdy's centrifugal method 274 

Removal of albumin 2 74 

Serum-globulin . . 274 

Qualitative tests 275 

Quantitative method 276 

Proteoses 276 

Primary proteoses 276 

Bence- Jones' protein 276 

Secondary proteoses . . 278 

Tests 279 

Bang's method 279 

Clinical significance 279 

Peptone 281 

Hemoglobin 281 

Heller's test 282 

Donogany's test 282 

Fibrin 283 

Carbohydrates 283 

Glucose 284 

Glycosuria 284 

Qualitative tests 288 

Trommer's test 288 



Xxiv TABLE OF CONTENTS. 

Page 

Hyaline casts . 356 

Granular casts 357 

Waxy casts 358 

Fibrinous casts 359 

Epithelial casts 359 

Fatty casts 359 

Blood-casts 359 

Pus-casts 359 

Cylindroids 360 

Pseudo-casts 361 

Cylindruria 361 

Spermatozoa 363 

Tissue fragments 363 

Bacteria 363 

Bacilluria 366 

Parasites 366 

V. Calculi 367 

Heller's table for analysis 368 

Uric acid calculi 368 

Calcium oxalate calculi 368 

Phosphatic calculi 368 

Calcium carbonate calculi 370 

Cystin calculi 370 

Xanthin calculi 370 

Urostealith calculi 370 

VI. Functional Diagnosis 370 

Cryoscopy 371 

Electric conductivity 371 

Chlorid excretion 372 

Methylene blue test 372 

Phloridzin test 373 

Phenolsulphonephthalein test 373 

CHAPTER VIL 
Secretions of the Genital Organs. 

I. Male Secretions . 376 

General considerations 376 

Microscopic examination 376 

Pathologic variations 378 

Medico-legal aspects 378 

• Florence's test 379 

Barberio's test 379 

II. Female Secretions 380 

Vaginal secretions 380 

Microscopic examination 380 

Pathology 381 

Blenorrhea 381 

Purulent secretions 382 

Fetid secretions 382 

Uterine secretions 382 

Menstruation 382 

The lochia 383 



TABLE OF CONTENTS. XXV 

Page 

Amniotic fluid 383 

Abortion 384 

Vesicular mole 384 

Carcinoma 384 



CHAPTER VIII. 

The Blood. 

I. General Considerations 385 

II. Physiology and Chemistry 386 

Blood formation and blood-forming organs 386 

Total volume of blood 387 

Volume relations of cells to plasma 390 

Methods of obtaining blood 392 

Physical properties 393 

Color 394 

Odor 394 

Reaction 395 

Specific gravity 399 

Viscosity 401 

Coagulation 401 

Osmotic pressure and cryoscopy 405 

Electric conductivity 406 

Chemical properties 407 

Total solids 409 

Blood pigments 409 

Hemoglobin 409 

Pseudo-hemoglobin 411 

Oxy-hemoglobin 411 

Met-hemoglobin 411 

Carbon monoxid-hemoglobin 412 

Carbon-dioxid-hemoglobin . .... 412 

Sulph-hemoglobin 412 

Decomposition products 413 

Hematin 413 

Hematoporphyrin 414 

Hematoidin 414 

Hemosiderin 415 

Malarial pigment 415 

Estimation of hemoglobin . 415 

Direct methods 416 

Indirect methods ...416 

Hemometer of Fleischl-Miescher 417 

Hemoglobinometer of Dare 420 

Hemometer of Sahli 421 

Hemoglobinometer of Oliver 422 

Hemoglobinometer of Tallqvist 424 

Variations in amount of hemoglobin 425 

Oligo-chromemia . . 426 

Color index 426 

Proteins of the blood 427 

Other nitrogenous constituents . .... . 430 

Total nitrogen 430 



XXVI TABLE OF CONTENTS. 

Page 

Total non-protein nitrogen 430 

Urea 435 

Uric acid 439 

Ammonia 443 

Creatinin 444 

Amino-acids 445 

Carbohydrates 445 

Fats and fatty acids 449 

Acetone 451 

Biliary constituents 451 

Inorganic constituents 452 

Blood gases 454 

Ferments of the blood 454 

Enumeration of the cells 455 

Hemocytometer of Thoma-Zeiss 456 

Hemocytometer of Durham 467 

Hemocytometer of Oliver 468 

III. Morphology of the Blood * ■ 469 

Examination of fresh blood 470 

Preparation of smears 471 

Fixation of smears 473 

Staining methods 476 

Erythrocytes 486 

Appearance and structure 486 

Size and shape 488 

Nucleation 490 

Number 492 

Normal variations 492 

Pathological variations 495 

Oligo-cythemia 495 

Poly-cythemia 495 

Staining properties 496 

Degenerations 498 

Isotonicity and resistance 499 

Variations in childhood and old age. 500 

Functions 501 

Leucocytes 502 

Appearance 502 

Types in normal blood 502 

Lymphocytes 502 

Large mononuclears 503 

Polymorphonuclear neutrophiles 504 

Polymorphonuclear eosinophiles 506 

Polymorphonuclear basophiles 506 

Types in pathological blood 507 

Myelocytes 507 

Irritation forms . 509 

Degeneration forms 509 

Differential counting 510 

Number 511 

Leucocytosis 512 

Physiological 513 

Pathological 515 

Mixed leucocytosis 518 



TABLE OF CONTENTS. XXV11 

Page 

Lymphocytosis 518 

Eosinophilia 519 

Mast-cell type 521 

Leucopenia 521 

Variations in infancy and childhood 521 

Functions 522 

Blood-plates 523 

Appearance 523 

Size. . .' 524 

Number 524 

Staining properties 524 

Function . . 525 

Hemoconien 525 

Morphology of the blood-forming organs 525 

IV, Pathology of the Blood 528 

Special 528 

Anemia 528 

Primary 528 

Simple primary anemia 528 

Chlorosis 529 

Progressive pernicious anemia 531 

Splenic anemia 532 

Anemia infantum pseudo-leukemica 533 

Leukanemia 534 

Aplastic anemia 534 

Secondary 534 

Acute hemorrhage 535 

Chronic hemorrhage 536 

Inanition 536 

Intestinal parasites 537 

Fever 538 

Blood poisons ' . . . 538 

Leukemia 539 

Spleno-myelogenous type 539 

Lymphatic type 543 

Acute type 543 

Pseudo-leukemia 544 

Hodgkin's disease 544 

Tuberculosis of the lymph glands 545 

Lympho-sarcoma . 545 

Gummatous lymphoma 545 

General 546 

Blood changes following surgical intervention 546 

Constitutional diseases 547 

Diabetes mellitus 547 

Gout 548 

Addison's disease 548 

Rickets 549 

Myxedema 549 

Acute infections 549 

Pneumonia 55° 

Typhoid fever 55 2 

Scarlet fever 553 

Measles 554 



XXV111 TABLE OF CONTENTS. 

Page 

Variola 555 

Diphtheria 556 

Pertussis 556 

Rheumatism 557 

Chronic infections 557 

Tuberculosis 557 

Syphilis 558 

Leprosy 559 

Carcinoma 559 

Effects of splenectomy 560 

V. Parasitology of the Blood 561 

Malaria 561 

Examination of fresh blood 563 

Tertian parasite 563 

Quartan parasite 565 

Estivo-autumnal parasite 566 

Stained specimens 568 

Sporogony 570 

General hematological changes 572 

Relapsing fever 574 

Sleeping sickness 575 

Kala-Azar 576 

Filariasis 577 

Syphilis 578 

Cultivation of treponema pallidum 581 

Yellow fever 583 

Rocky mountain spotted fever 584 

Distomiasis 585 

VI. Bacteriology of the Blood 585 

Technic 585 

Organisms found in the blood 587 

VII. Serum Pathology 593 

Ehrlich's side-chain theory 594 

Phagocytosis 598 

Opsonins 598 

Diagnostic use of vaccines 600 

Tuberculin reactions 601 

Luetin reactions 603 

Schick reaction 605 

VIII. Sero-Diagnosis 606 

Agglutination reactions 607 

Gruber-Widal test 607 

Method of Bass and Watkins 611 

Diseases other than typhoid 613 

Precipitin reaction 614 

Complement-fixation test 617 

Wassermann reaction 620 

Noguchi method 629 

Diseases other than syphilis 636 

Abderhalden's Sero-Diagnosis. 638 

Of pregnancy 638 

Of other conditions 649 

Herman-Perutz reaction 651 

Tests before transfusion 653 



TABLE OF CONTENTS. XXIX 

Page 

IX. Medico-legal Aspects 654 

Red cells 654 

Guaiac test 655 

Schaer's test 655 

Phenolphthalin test 656 

Teichmann's test 657 

Spectroscopic examination. . 658 

Precipitin test 658 

X. Value and Limitations of Blood Examinations 659 



CHAPTER IX. 

Transudates and Exudates. 

I. General Considerations 662 

II. Physical and Chemical Properties 663 

Serous exudates 664 

Chylous exudates 665 

Chyloid exudates 665 

Hemorrhagic exudates 665 

Purulent exudates 666 

Putrid exudates 666 

III. Bacteriology 667 

Tubercle bacilli 667 

Inoscopy 667 

Gonococci 667 

Smegma bacilli 669 

Ducrey's bacilli 669 

Spirochete pallidas 669 

IV. Cytology 672 

Technic 672 

Cytology of normal fluids ; . . . 673 

Cytology of pathological fluids 673 

Pleural exudates -.. 674 

Primary tubercular pleurisy 674 

Secondary tubercular pleurisy 674 

Pneumococcus pleurisy 674 

Streptococcus pleurisy 675 

Typhoid pleurisy 675 

Malignant pleurisy 675 

Nephritic and cardiac pleurisy .-..'.''. : 675 

Peritoneal exudates 675 

V. Cyst Fluids 676 

Ovarian cysts 676 

Serous cysts 676 

Myxoid or colloid cysts 676 

Papillary cysts 677 

Dermoid cysts 677 

• Parovarian cysts 677 

Hydrocele 677 

Spermatocele 678 

Hydronephrosis 678 

Hydatid cysts 678 

Pancreatic cysts 678 



XXX TABLE OF CONTENTS. 

Page 

VI. Cerebrospinal Fluid 678 

Lumbar puncture 679 

Microscopic examination 682 

Epidemic cerebrospinal meningitis 682 

Tubercular meningitis 684 

Acute anterior poliomyelitis 684 

Cerebrospinal syphilis 685 

Noguchi's butyric acid test 685 

Nonne's test 685 

Ross-Jones test 686 

Lange's colloidal-gold test 686 



CHAPTER X. 

Secretion of the Mammary Glands. 

I. General Considerations 688 

II. Physical and Chemical Properties 689 

Appearance and color 690 

Specific gravity 690 

Reaction 691 

Coagulation 691 

Total solids 691 

Ash 691 

Protein 691 

Total protein 692 

Method of Sebelien 692 

Method of Boggs 692 

Casein 693 

Albumin and globulin 693 

Fat 693 

Babcock's method 693 

Extraction method 694 

Lactose 695 

Preservatives in cow's milk 695 

Sodium carbonate 695 

Salicylic acid 696 

Formaldehyde 696 

Boric acid and borax 696 

III. Bacteriological Examination of Milk 696 



CHAPTER XI. 

Clinical Bacteriology. 

I. General Considerations 699 

II. Sterilization 700 

III. Preparation of Culture Media 701 

IV. Incubation 704 

V. Preparation of Cultures 705 

VI. Staining ' 707 

VII. Identification of Organisms 709 



TABLE OF CONTENTS. XXxi 

Page 
VIII. Vaccines * 723 

Preparation of vaccines 724 

Stock vaccines 724 

Mixed vaccines 725 

Autogenous vaccines . 725 

Anti-typhoid vaccination. 726 

Index 728 



LIST OF ILLUSTRATIONS. 



PLATES. 

To Face Page 

I. Tubercle Bacilli in Sputum 20 

II. Streptococcus Pyogenes 26 

III. Leptothrix and Spirocheta Buccalis (Unstained) 35 

IV. Diphtheria Bacilli Showing Polar Staining 39 

V. Endamebae Gingivalis (Gros.) 42 

VI. Koch-Weeks Bacillus 47 

Morax-Axenfeld Diplobacillus 47 

VII. Trachoma Bodies of Prowazek-Greeff 48 

VIII. Vegetable Cells found in Feces 155 

IX. Osazons 294 

X. Ammonium Urate Crystals 347 

XI. Waxy Casts 359 

XII. Mucous Threads in Urine (Unstained) 361 

XIII. Cystitis Due to Colon Bacillus 366 

XIV. Staphylococcus Cystitis 367 

XV. Absorption Spectra 4 IQ 

XVI. Absorption Spectra 4 11 

XVII. Fresh Normal Blood , 47° 

XVIII. Types of Red Cells 488 

XIX. Ring Bodies in Red Cells 49 8 

XX. The Leucocytes 5° 2 

XXI. Iodophilia 5°9 

XXII. Polynuclear Leucocytosis 5 12 

XXIII. Chlorotic Anemia ,. . 5 2 9 

XXIV. Blood in Pernicious Anemia 53 1 

XXV. Blood in Leukanemia 534 

XXVI. Blood in Spleno-myelogenous Leukemia 54° 

XXVII. Lymphatic Leukemia 543 

XXVIII. The Tertian Parasite (Unstained) 5 6 3 

XXIX. The Quartan Parasite (Unstained) 5 6 5 

XXX. The Estivo-autumnal Parasite (Unstained) 5 6 7 

XXXI. Tertian Parasite (Stained) 5 68 

XXXII. Estivo-autumnal Parasite (Stained) 57° 

XXXIII. Gonococci in Urethral Discharge 66 S 

XXXIV. Spirochete Pallidas in Tissue , 670 

xxxiii 



XXXIV LIST OF ILLUSTRATIONS. 

To Face Page 

XXXV. Exudate from Tubercular Pleurisy . 674 

XXXVI. Exudate in Pneumonic Pleurisy 675 

XXXVII. Exudate in Malignant Pleurisy 676 



FIGURES. 



Page 

i. Curschmann's Spirals 7 

2. Objects Found in the Sputum 10 

3. Aspergillus Fumigatus 15 

4. Micrococcus Catarrhalis 15 

5. Budding Forms of Blastomycetes 16 

6. Diplococcus Pneumoniae 25 

7. Friedlander's Bacillus 25 

8. Bacillus Influenzae 26 

9. Actinomyces 28 

10. Paragonimus Westermanii 29 

11. Ovum of Paragonimus Westermanii 30 

12. Vincent's Spirillum and Bacillus 40 

13. Oidium Albicans 41 

14. Stomach Tube 52 

15. Turck's Aspiration Apparatus 53 

16. Boas-Oppler Bacillus 63 

17. Strauss' Separatory Funnel 76 

18. Sahli's Desmoid Bag 88 

19. Normal Feces 100 

20. Boas' Stool-Sieve 104 

21. Schmidt's Fermentation Apparatus 124 

22. Cholera Spirilla 129 

23. Bacillus Typhosus 130 

24. Amoeba Coli 135 

25. Coccidium Hominis . , 136 

26. Trichomonas Intestinalis . 137 

27. Cercomonas Hominis 138 

28. Megastoma Entericum 138 

29. Balantidium Coli 139 

30. Taenia Solium 140 

31. Taenia Saginata 141 

32. Taenia Cucumerina 141 

33. Taenia Nana 142 

34. Taenia Diminuta 142 

35. Ovum of Taenia Diminuta 142 

36. Taenia Echinococcus 143 



LIST OF ILLUSTRATIONS. XXXV 

Pagb 

37. Hydatid Cyst I43 

38. Bothriocephalic Latus 144 

39. Dibothriocephalus Cordatus 144 

40. Ascaris Lumbricoides . ... I4 ^ 

41. Ascaris Mystax I4 6 

42. Oxyuris Vermicularis I4 y 

43. Strongyloides Intestinalis 148 

44. Trichiuris Trichura 149 

45. Trichinella Spiralis x^ 

46. Tail of Uncinaria Duodenalis 151 

47. Anterior End of Uncinaria Duodenalis ...151 

48. Tail of Uncinaria Americana 153 

49. Anterior End of Uncinaria Americana 153 

50. Parasitic Bodies, Ova, and Larvae 154 

51. Fasciola Hepatica 157 

52. Fasciolopsis Buski 158 

53. Opisthorchis Felineus 159 

54. Opisthorchis Sinensis 160 

55. Eustrongylus Gigas 161 

56. Acarus Scabiei 162 

57. Demodex Folliculorum 163 

58. Leptus Autumnalis 163 

59. Pediculus Capitis . 164 

60. Pediculus Vestimenti 164 

61. Pediculus Pubis 164 

62. Pulex Irritans 165 

63. Pulex Penetrans 166 

64. Achorion Schonleinii 167 

65. Normal Hair 168 

66. Trichophyton Endo-ectothrix 168 

67. Microsporon Audouini 169 

68. Mycelial Threads of Blastomycetes 170 

69. Budding Forms of Sporothrix Schenckii 171 

70. Urinometer and Cylinder 188 

71. Volumetric Flasks 203 

72. Kjeldahl's Nitrogen Apparatus 218 

73. Doremus' Ureometer 222 

74. Doremus-Hinds Ureometer 223 

75. Folin's Urea Apparatus 224 

76. Schlosing's Ammonia Apparatus 229 

77. Folin's Ammonia Apparatus 230 

78. Folin's Absorption Bulb 231 

79. Ruhemann's Uricometer 239 

80. Sargent's Colorimeter 244 

81. Conical Test-glass 269 

82. Horismascope 269 



XXX VI LIST OF ILLUSTRATIONS. 

Page 

83. Esbach's Albuminometer 273 

84. Laurent Polariscope 300 

85. Diagrammatic Representation of the Course of Light through the 

Laurent Polariscope 301 

86. Einhorn's Saccharometer 202 

87. Lohnstein's Fermentation Tube for Undiluted Urine 304 

88. Lohnstein's Fermentation Tube for Diluted Urine 304 

89. Purdy Electric Centrifuge 336 

90. Sediment Tube 337 

91. Percentage Centrifuge Tube 337 

92. Various Forms of Uric Acid 339 

93. Acid Sodium Urate 340 

94. Xanthin 340 

95. Calcium Oxalate 341 

96. Cystin 342 

97. Pure Leucin. . 343 

98. Impure Leucin . 344 

99. Tyrosin 344 

100. Calcium Sulphate 345 

101. Bilirubin 345 

102. Cholesterin 347 

103. Magnesium-Ammonium Phosphate 348 

104. Calcium Carbonate • 349 

105. Urinary Epithelium 350 

106. Pus Corpuscles 352 

107. Hyaline Casts 356 

108. Granular Casts 358 

109. Epithelial Casts 359 

no. Fatty Casts 360 

in. Blood, Pus, Hyaline, and Epithelial Casts 361 

112. Cylindroids 362 

113. Scolex and Hooklets of Taenia Echinococcus in Urine 366 

114. Ova and Miracidium of Schistosomum Hematobium . - 367 

115. Normal Semen 377 

116. Chorionic Villi 384 

117. Daland's Hematocrit 390 

118. Hematocrit Tube 391 

119. Blood Needle 393 

120. Dare's Hemoalkalimeter 396 

121. Pycnometer 400 

122. Boggs' Coagulometer 404 

123. Beckmann Apparatus 405 

124. Direct-vision Spectroscope ' 410 

125. Hemometer of Fleischl-Miescher 417 

126. Hemoglobinometer of Dare . 420 

127. Method of Filling the Dare Blood Pipet 421 



LIST OF ILLUSTRATIONS. XXXvii 

Page 

128. Hemometer of Sahli 422 

129. Hemoglobinometer of Oliver 423 

130. Tallqvist's Hemoglobinometer 424 

131. Thoma-Zeiss Counting Chamber 456 

132. Diluting Pipets 457 

133. Ruled Surface of Thoma-Zeiss Counting Chamber 458 

134. Turk's Ruling of Counting Chamber 458 

135. Plan of Counting the Cells 464 

136. Cross-section of Durham's Blood Pipet 467 

137. Oliver's Hemocytometer 468 

138. Preparation of Blood-smears with Glass Slides 471 

139. Preparation of Blood-smears with Cigarette Paper 472 

140. Ehrlich Forceps 472 

141. Pinch Forceps 473 

142. Oven for Fixing Blood-films 474 

143. Normal Blood Showing Rouleaux Formation and Fibrin Network . 486 

144. Cycles of the Malarial Parasite 571 

145. Spirillum of Obermeier 574 

146. Trypanosoma Gambiense 575 

147. Filaria Bancrofti 577 

148. Spirochetal Pallidas and Refringens 580 

149. Ultra-condenser of Reichert 581 

150. Schistosomum Hematobium 584 

151. Illustrating the Mechanism of Toxin-cell Union 594 

152. Illustrating the Elaboration and Action of Antitoxin . 595 

153. Illustrating the Mechanism of Hemolysis 596 

154. Illustrating the Mechanism of Antihemolysis 597 

155. Bacillus Typhosus at Beginning of Widal Test 607 

156. A Pseudo- Widal Reaction 607 

157. A Positive Widal Reaction 608 

158. Hemin Crystals from Human Blood . . . 657 

159. Lumbar Puncture 680 

160. Diplococcus Intracellularis Meningitidis 683 

161. Normal Milk and Colostrum 688 

162. Babcock Milk and Cream Bottles . 694 

163. Bottle for Human Milk 694 

164. Soxhlet Apparatus 695 

165. Wolfhugel's Colony Counter 697 

166. Lautenschlager Hot Air Sterilizer 699 

167. Autoclave 700 

168. Arnold Sterilizer 701 

169. Incubator 705 

170. Platinum Needles 706 

171. Apparatus for Anaerobic Cultivation 707 



DIAGNOSTIC METHODS. 



CHAPTER I. 

THE SPUTUM. 

I. General Considerations. 

At the present time the examination of the sputum is more or less lim- 
ited to the search for various specific organisms, especially the bacilli of tuber- 
culosis and of pneumonia. This is very much to be regretted, as frequently the 
appearance, amount, consistency, color, and other characteristics are of great 
aid and have led our older brothers to correct diagnoses before the days of 
microscopic examination. 1 

The sputum, strictly speaking, should be considered as the material which 
comes from the respiratory passage anywhere along its course. It may be, 
therefore, of laryngeal, bronchial, or alveolar origin. More commonly, how- 
ever, we find the sputum considered by the general practitioner and by the 
laity as anything which is expectorated, so that specimens which consist of 
nothing more than salivary secretion are frequently sent to laboratories for 
examination. In some cases, especially of inflammation of the naso-pharynx 
or perforation from neighboring organs, this buccal secretion may be mixed 
with material from the nose, mouth, ear, or esophagus. For these reasons, 
if for no others, it is absolutely essential that frequent examinations of sputum, 
in the strict sense, be made before a negative diagnosis of a suspected condition 
may be given. 

As a rule, any sputum at all should be considered pathological, as normal 
persons raise little or nothing from the lungs at any time. By this is not meant 
that serious disease may obtain when a small amount of sputum exists, as 
patients suffering with catarrhal conditions of the naso-pharynx frequently have 
an accumulation of material, which has settled in the bronchial tubes over night 
and is raised in the morning. Those of us who are unfortunate enough to live 
in atmospheres which are loaded with soot and dirt frequently raise a certain 
amount of sputum which arises from an increased activity of the mucous mem- 
brane of the respiratory passages to compensate for the dryness and irritation 
which these foreign substances have caused. This morning sputum is small 
in amount and is in the form of large, tough, elastic masses which very much 
resemble boiled sago. These masses are at times extremely dark in color, due 
to the dirt which has been taken in with the inhaled air. In such sputum we 
find much mucus, degenerated epithelium, pus cells and various micro-organ - 

1 See Laird, Jour. Am. Med. Assn., 1915, LXIV, 427. 



2 DIAGNOSTIC METHODS. 

isms. These organisms are rarely of pathologic significance, although many 
pathogenic types may be present. 

The sputum should be collected in receptacles which may be completely 
and easily disinfected. The best receivers for the sputum are the ordinary paper 
spit-cups which can be burned as occasion may demand. The practice of ex- 
pectorating upon cloths is only to be advised when these cloths are immediately 
burned. In the spit-cup should always be contained a certain amount of disin- 
fectant, such as dilute carbolic acid or corrosive sublimate solution, so that no 
chance of transference of infection may arise. Neglect of such precautions 
has frequently led to serious consequences in the case of the healthy members 
of the family. When sputum is to be collected for examination, the material 
raised by coughing should be received in a wide-mouthed bottle which contains 
no disinfectants, as these agents coagulate the protein material of the infecting 
organism and in some cases change its staining characteristics. Immediately 
after examination of such material it should, of course, be treated with the 
disinfecting solution. 

II. Physical and Chemical Characteristics. 
Amount. 

Some general idea of the amount of sputum expectorated is always advis- 
able. It is rarely necessary, however, to make any collection of the material 
during the 24 hours' period, as one can usually gain the information by question- 
ing the patient or nurse as to the amount passed from time to time. The 
amount of sputum expectorated in 24 hours depends very much upon the nature 
of the pathologic condition. In cases of so-called dry bronchitis, diffuse 
bronchitis, early tuberculosis, and occasionally of lobar pneumonia, the spu- 
tum is so viscid that there is practically none obtained. In cases of chronic 
bronchitis, tuberculosis with cavity formation, and bronchiectasis we find large 
amounts; while in cases of lung abscess or of perforating pleurisy, blood or pus 
may flow from the mouth in very large quantities. The absolute amount of 
sputum may, therefore, vary from a cubic centimeter to a liter or more. An 
extensive expectoration will naturally have more or less serious effect upon the 
patient's general nutrition, so that we are not surprised to find cases in which 
as high as 5 per cent, of the total nitrogen eliminated passes from the system 
in the sputum (Lenz). 

Consistency. 

As a rule, the consistency of the sputum varies from that of a liquid to a 
highly tenacious material, inversely as the amount of sputum. This latter 
statement holds in most cases with the exception of pneumonia, in which we 
have an extremely tenacious sputum and, also, a very abundant one. Just 
what substance induces this extreme tenacity is uncertain, but it may be gener- 
ally said that mucin is the causative factor, although the tenacious pneumonic 
sputum shows very little mucin. According to Kossel, the tenacity may be 
due to the presence of nuclein derivatives. In the early stages of acute bron- 
chitis, or bronchial asthma, and in whooping-cough the sputum is usually very 



THE SPUTUM. 3 

tenacious and ropy; while in edema of the lungs, pulmonary abscess, putrid 
bronchitis, and pulmonary gangrene it is very watery and contains large num- 
bers of pus-cells. 

Reaction. 

The fresh specimen of sputum is usually alkaline in reaction. However, 
in cases in which the sputum has remained in the lungs for some time, as in 
cavity formation, the reaction is acid. 

Color. 

The color of the sputum may range from that of a colorless material to one 
showing any of the tints of the rainbow. These colors are due to admixtures 
of various abnormal products with the sputum. A bright red sputum is sig- 
nificant of the presence of blood, according to Traube, unchanged red blood- 
cells necessarily being present. The amount of blood may vary from a light 
streaking of the sputum to one showing a deep red, rusty, or prune-juice color. 
Such bloody sputa are found after trauma, pneumonia, gangrene, hemorrhagic 
infarction of the lungs, chronic passive congestion, as well as adventitious mix- 
ture of sputum with nasal or pharyngeal material containing blood. The 
blood may be due to rupture of a vessel and may then constitute the condition 
known as pulmonary hemorrhage. This condition of hemoptysis 1 is frequently 
confounded with that of hematemesis and is differentiated by the fact that the 
blood in hemoptysis is frothy, bright red in color, alkaline in reaction, and usu- 
ally associated with mucopurulent material; while the blood in hematemesis is 
frequently dark and grumous, usually clotted and acid in reaction. In sputa 
which are tinted by changed hemoglobin, the color is most varied. Many 
oxidation products of hemoglobin are found in the sputum, depending upon 
the time which the sputum has lain in the lungs. Thus, for instance, in pneu- 
monia we find a rusty, prune-juice colored sputum, whose color seems to be due 
to an unknown derivative of hemoglobin. The shade of color in pneumonia 
may, however, range from red through brown to green. In cases of mitral 
disease associated with passive congestion of the lungs we find, frequently, 
a light brown color, due to the presence of hematoidin granules in the 
epithelial cells. 

In many cases of abscess of the liver which perforates into the lung or 
in catarrhal jaundice, bile pigments may be found in the sputum which may give 
rise to various colored sputa from red through blue to green. Although, as 
shown in a later table, the bile pigments and hemoglobin are very closely re- 
lated chemically, yet the clinical significance of the appearance of bile pigment 
in such cases as the above is very important. In many cases of true jaundice 
the sputum, in case any exists, may show a distinct grass-green color due to 
the presence of oxidized bile pigments. This same green color frequently 
appears in the sputum, however, in cases of croupous pneumonia during lysis. 
In this latter case the color is due, probably, to the same pigment which is oxi- 
dized before expectoration. In these cases of pneumonia with green sputum 
and no jaundice, a fresh involvement of the lung is usually associated with a 
1 See van der Hoeven-Leonhard, Arch, internat. de laryngol., 1913, XXXV, 831. 



4 DIAGNOSTIC METHODS. 

rusty sputum. Besides jaundice and pneumonia we may have, as causes 
of green sputum, certain chloromata of the lungs as well as the development of 
chromogenic bacteria within the lungs. This action of chromogenic bacteria 
is not always observed when the sputum is expectorated, but may appear only 
after the sputum has stood. The presence of the bacillus pyocyaneus may 
give a sputum which is brilliant blue or greenish in tint. 

Sputa very frequently show changes in color as well as consistency due 
to various substances inhaled. Thus we find a distinctly black sputum, in 
cases of anthracosis, in coal-miners and in many city residents. So frequent 
is this occurrence that the lung tissue may even be invaded by the coal pigment. l 
Workers in bronze and brass as well as other metals frequently show a spu- 
tum tinged red with ferric oxide, arising from a condition of the lung known as 
siderosis. 2 Stone-cutters frequently show much stone-dust in the sputum which 
is characteristic of the condition of chalicosis, stone-cutters' phthisis, or grinders' 
rot. Workers in flour mills and in bakeries frequently expectorate doughy 
masses, while those in cotton mills show the presence of cotton fibers in their 
sputum. Finally it may be said that the color of the sputum may be changed by 
the presence of certain foods, such as milk, eggs, and chocolate, while tobacco 
users frequently have a sputum tinged dark brown. 

Odor. 

Ordinarily the sputum has no odor unless it has stagnated either in the 
receiving cup or in the system. Such old sputum sometimes has a very distinct 
putrefactive odor. The odor of sputum in tuberculosis and bronchiectasis is 
peculiarly heavy and sweet, while that in putrid bronchitis is often extremely 
offensive. In cases of perforating empyema a peculiar cheese-like odor is 
observed. While these odors are not characteristic in themselves, they are 
usually more or less significant of the condition with which they are commonly 
associated. 

Character of the Sputum. 

The character of the sputum has reference more to the apparent com- 
position of the sputum than to its consistency. Air is usually present in the 
sputum in various amounts, so that one may judge, from the size of the air- 
bubbles, of the size of the bronchi from which the sputum came. The sputum 
from cavities and large bronchi contains no air and, therefore, sinks in water. 
This is the so-called sputum fundum petens. 

Sputum known as mucoid sputum is glairy, transparent, and tenacious, 
becoming cloudy on the addition of acetic acid due to the precipitation of mucin. 
This type of sputum is found particularly in acute bronchitis and in asthma. 

A mucopurulent sputum is one containing both pus and mucoid material. 
Small amounts of pus give a whitish color, either to the whole or to portions of 
the sputum, the pus being observed in masses or in streaks through the mucoid 
material. Larger quantities of pus give a yellowish or occasionally a yellowish- 
green tinge to the sputum. In this latter type the pus and mucus seem to be 

1 See'Fiiadoro, Med. nuova, 1913, IV, 133; also, Haythorn, Jour. Med. Research, 1913, 
XXIX, 259; Klotz, Am. Jour. Pub. Health, 1914, IV, 887. 

2 See Gigon, Beitr. z. path. Anat. u. z. allg. Path., 1912, LV, 46. 



THE SPUTUM. 5 

mixed homogeneously. In the sputum from cavities we find the mucopuru- 
lent material arranging itself flatly like a coin, constituting the so-called u num- 
mular" sputum. 

Purulent sputum is found in cases of ruptured empyema, abscess of the 
lung, and in some cases of bronchiectasis. This purulent sputum differs from 
the mucopurulent type in the fact that the pus is much more abundant and is 
almost in the pure state, being mixed with a small amount of tenacious mucus. 

In some cases, especially in edema of the lungs, a sputum is obtained, known 
as serous sputum, which is colorless and quite frothy. This sputum resembles 
very closely the ordinary salivary secretions and should not be confused with it. 

In many cases, especially in putrid bronchitis, gangrene of the lung, and 
bronchiectasis, the sputum on being voided into a cylinder will separate into 
three distinct layers, occasionally into four. The upper layer is of frothy 
mucus; a second layer, which is not always present, consists of certain albumin- 
ous material which hangs in long shreds down into the third layer, which con- 
sists of the sero-pus and is usually opaque and watery. The bottom layer 
contains the morphological elements, pus, tissue shreds, and bacteria. 

Besides the varieties of sputum named above, the admixture of blood may 
give rise to sputum which is known as sanguinous sputum or as sanguino-muco- 
purulent or purulent sputum. 

Chemical Properties. 

The chief chemical examination is applied to the detection and estimation 
of the amount of albumin and of mucin in the sputum. The method is as 
follows: 1 Treat the sputum with an equal quantity of 3 per cent, acetic acid, 
in order to precipitate the mucin. Shake thoroughly for a few minutes and allow 
to stand for 15 minutes. Filter through filter-paper until a perfectly clear 
filtrate is obtained, adding a known amount of water to facilitate filtration if 
necessary. Apply to this filtrate any of the tests for albumin given in the 
section on urine. If the quantitative determination is desired, use Tsuchiya's 
reagent as the precipitant, multiplying the result by the dilution of the sputum 
to obtain the true percentage of albumin. 

A large amount of albumin indicates, of course, such conditions as pneu- 
monia, pulmonary edema or perforating empyema. While the early workers 
regarded anything more than a slight opalescence as pathological, a trace of 
albumin indicating a pulmonary tuberculosis in their opinion, later investiga- 
tors showed that albumin is present in the sputum whenever there is active 
exudation into the alveoli as well as into and under the pleura. Many more 
recent studies have proven that albumin may not be present in the sputum of 
true pulmonary tuberculosis, but is frequently seen in benign affections and 
when present is due, in the majority of cases, to occult blood. For these reasons 
it is more than probable that such chemical tests are to be given merely a pre- 
sumptive interpretation. 2 

1 Lesieur and Prires, Paris med., 19 n, IV, 29. 

2 See Wanner, Deutsch. Arch. f. klin. Med., 1903, LXXV, 347; Roger and Levy-Valensi, 
Presse med., 1910, XVIII, 289; Ibid., 1911, XIX, 409; Geeraerd, Jour, de med., de chir. et 
de pharmacol., 1910, XV, 505; Raymond, Presse med., 1911, XIX, 675; Goodman, Arch. Int. 
Med., 1911, VIII, 163; Fishberg and Felberbaum, Med. Record, 1911, LXXX, 870; Fishberg, 



6 DIAGNOSTIC METHODS. 

Besides these albuminous principles, the sputum, especially in cases of 
gangrene and putrid bronchitis, contains a ferment very much resembling in 
its action the trypsin of the pancreatic juice (Stolnikow). 1 This ferment seems 
to indicate a highly destructive process in the lung tissue. Other chemical, 
substances, such as glycogen and fatty acids, 2 are frequently found in the sputum 
but may be passed with mere mention. The so-called myelin granules or 
globules, which appear in the alveolar cells of the sputum, consist largely of 
fatty principles, such as protagon, lecithin and cholesterin. 

III. Macroscopic Examination. 

While much that is included in the previous section would come properly 
under the head of the macroscopic examination of the sputum, the writer has 
reference more, in this connection, to the appearance of macroscopic elements 
as distinguished from those which are purely microscopic. 

(i). Cheesy Masses. 

Frequently one finds in the sputum small cheese-like particles which 
vary in size from that of a pin-point to that of a pea, the large majority being 
about the limit of ordinary vision. These cheesy masses are fragments of ne- 
crotic tissue and appear in the larger form in cases of abscess or gangrene of the 
lung, while in tuberculosis they are always small unless the cavity, from which 
the material is derived, is markedly necrotic. The color of these masses varies 
from a yellow to a black. Those fragments which come from an abscess are of 
yellow color due to the presence of much pus, the darker ones contain decompo- 
sition products of hemoglobin, while many of them are tinged a deep black with 
coal pigment. If the sputum be squeezed between two glass plates these cheesy 
particles or fragments can sometimes be more distinctly seen. They are pres- 
ent in largest numbers in the so-called "nummular" sputum from a tubercu- 
lous cavity. 

(2). Dittrich's Plugs. 

These masses are similar to the small caseous particles above mentioned 
and are frequently expectorated by perfectly normal individuals. The true 
plugs are distinct casts of the bronchi or bronchioles and vary in size from 

Arch. Diagnosis, 1912, V, 220; Works, Jour. Am. Med. Assn., 1912, LIX, 1537; Scott, Ibid., 

1913, LX, 440; Acs-Nagy, Wien. klin. Wchnschr., i9i2,XXV, 1904; Kauffmann, Beitr. z. Klin, 
d. Tuberk., 1913, XXVI, 269; Schmitz, Med. Klin., 1913, IX, 1163; Berkovits and Rudas, Berl. 
klin. Wchnschr., 1913, L, 1752; Ridge and Treadgold, Lancet, 1913, II, 382; Schneider, 
Zentralbl. f. inn. Med., 1913, XXXTV, 1025; Gelderblom, Deutsch. med. Wchnschr., 1913, 
XXXIX, 1987; Maliwa, Deutsch. Arch. f. klin. Med., 1913, CXII, 231; Hohn and Himmel- 
berger, Jour. Am. Med. Assn., 1914, LXII, 20; Lewis, New York Med. Jour., 1914, XCLX, 
1031; Melikjanz, Med. Obozr., 1914, LXXX, 819; Beitr. z. Klin. d. Tuberk., 1914, XXX, 81; 
Wien. klin. Wchnschr., 1914, XXVII, 653; Cocke, Am. Jour. Med. Sc, 1914, CXLVIII, 724; 
Hafemann, Deutsch. med. Wchnschr., 1914, XL, 1715; Benzler, Beitr. z. Klin. d. Tuberk., 

1914, XXXII, 363; Glover, Brit. Jour. Tuberc, 1914, VIII, 217; Holm and Chambers, Jour. 
Michigan Med. Soc, 1914, XIII, 226; Callender, Med. Record, 1914, LXXXVE, 783; Fantoni, 
Gazz. d. osp., 1914, XXXV, 417; Lowenbein, Ztschr. f. Tuberk., 1915, XXIII, 122; Szaboky, 
Ibid., 352; Snijders, Nederl. Tijdschr. v. Geneesk., 1915, I, 349; Lockwood, Jour. Am. Med. 
Assn., 1915, LXIV, 574. 

1 See Eiselt, Ztschr. f. klin. Med., 1912, LXXV, 71; also, Maliwa, Deutsch. Arch. f. klin. 
Med., 19 14, CXV, 407. 

2 See Barbaro, Gazz. d. osp., 1915, XXXV, 985. 



THE SPUTUM. 7 

pin-point to that of a bean, the majority being about the size of a mustard seed. 
The smaller ones are opaque and yellowish-white in color, while the larger 
ones have a distinct gray tinge. They are usually expectorated free from pus 
or mucus, so that they frequently give rise to anxiety, especially in those of a 
hypochondriac tendency. These plugs have a distinctly disagreeable odor, 
which is more evident if they are crushed on the glass plates. Microscopic 
examination of these masses shows large clumps of bacteria, fatty acid crystals, 
free fat globules, and cellular detritus. Occasionally a few leucocytes are found, 
but these are rare, while pigment granules, either of hematogenous or extraneous 
origin, are sometimes observed. While such plugs are especially numerous 
in cases of putrid bronchitis and bronchiectasis, they are frequently found in the 
crypts of the normal tonsil as well as in cases of follicular tonsillitis or of ozena. 






I. II. III. 

Fig. i. — Curschmann's spirals {Tyson after Curschmann) . I, Natural size; 
II and III, enlarged; a, a, central thread. 



(3). Curschmann's Spirals. 

These structures are found in the sputum in practically every case of 
true bronchial asthma and have been reported in acute bronchitis, croupous 
pneumonia, chronic pulmonary tuberculosis, and occasionally in chronic 
bronchitis. They are not present in every paroxysm of asthma, but are more 
frequently found just at the end of the paroxysm and are absent when the spu- 
tum becomes mucopurulent. They seem to be derived through true exuda- 
tion from the bronchioles, as Curschmann says, a bronchiolitis exudativa. 
These structures are recognizable, to a certain extent, by the naked eye, but for 
their absolute identification a microscopic examination is essential. They are 
composed of a spirally-twisted network of very delicate fibrils, in the meshes 
of which are numerous epithelial cells and eosinophile leucocytes. Along with 
these cellular bodies one frequently finds large numbers of diamond-shaped 
crystals, known as the Charcot-Leyden crystals, which will be discussed later. 
This spirally-twisted mass (the mantle) seems to be wound around a central 
light thread. While this is the structure of the complete spiral, we frequently 



8 DIAGNOSTIC METHODS. 

find variations pointing apparently to two distinct forms. The first is the 
spirally-twisted strand of mucus with the enclosures above mentioned. The 
second is the tight spiral mass of mucus wound around a central fibre. 
This central fibre is very refractive and is homogeneous in structure, varying in 
size from 1/2 to 18 microns in diameter. The length of these spirals is from 
1 to 2 cm. and their breadth about 1 mm. 

(4). Fibrinous Casts. 

By the term fibrinous cast we refer more directly to true bronchial casts, 
which are composed of fibrin. These are observed in pneumonia, in which case 
they are derived from the smaller bronchioles and are brownish or reddish in 
color and contain many red and white blood-cells. These smaller casts vary 
from 1/2 to 3 cm. in length. In the chronic fibrinous bronchitis we find the so- 
called arborescent casts which are usually whitish in color and contain many 
epithelial cells. These casts vary in size from 1 to 15 cm. in length by several 
mm. in thickness. We may have an acute form of fibrinous bronchitis accom- 
panying various febrile conditions, so that similar casts may appear in almost 
any of the infectious diseases. These larger casts are fairly firm, usually have 
a lumen, and branch dichotomously five to ten times. Microscopic examina- 
tion shows them to consist of large numbers of longitudinal fibres containing 
blood and epithelial cells in their meshes. They may be stained with the Wei- 
gert fibrin stain in a very beautiful way. Such staining methods show that not 
all of the material in such casts is fibrin, so that it may be necessary to rename 
these structures as simple bronchial casts rather than as fibrinous forms. 

Besides these fibrinous casts, one occasionally may find distinct bronchial 
casts composed of the mycelium of various fungi. Thus mycelial threads of 
the aspergillus have been reported by Osier, Devillers and Renon. 

(5). Concretions. 

This name is applied to anything, expectorated with the sputum, which 
has the appearance or consistency of a stone. These concretions are formed 
in dilated portions of the bronchi or in cavities by the calcification of the stag- 
nated contents. Although concretions consisting of cartilaginous or osseous 
material are frequently found postmortem, yet there seems to be little mention 
in the literature of any such formations being expectorated. 1 
(a). Bronchioliths. 

These concretions are formed by the deposition of calcium salts in the 
stagnated contents of the bronchus or of a cavity. They may be derived from 
the smaller or larger bronchi, but rarely form arborescent shapes, being usually 
irregular and varying in size from a pin-head to that of a walnut. They are 
usually single, but may be multiple. They vary in consistency from that of 
chalk to a stone, and may be expectorated in small numbers over long periods of 
time. 

(b). Pneumoliths. 

These lung stones are in the majority of cases of tuberculous origin. 
They usually arise by the calcification of caseous areas which later ulcerate into 
1 See Cade and Guerin, Lyon med., 19 14, XLVI, 221. 



THE SPUTUM. 9 

a bronchus and are expectorated unless too large. They may, also, arise by 
the calcification of a pulmonary cavity or of a bronchial lymph-gland. These 
lung stones consist of the carbonates, phosphates, and sulphates of calcium 
and magnesium, in some one or more salts predominating, while in others 
still different combinations may exist. These pneumoliths either have a 
chalky or a calcareous consistency and vary in size from that of a pin-head to 
that of a tennis-ball. These lung stones are usually expectorated en masse 
or in the form of smaller portions of a larger stone. In some cases these smaller 
stones may reach the number of 500 (Portal). 

(6). Echinococcus Membranes. 

Rarely one may find in the sputum fragments of the walls of echinococcus 
cysts or their contents. These may come from a perforating cyst of the liver, 
kidney, or lung. The presence of the laminated membrane and of the parasitic 
scolices and hooks makes it possible to arrive at an absolute diagnosis of the 
origin of such material. The membrane is thick, tough, and of a porcelain- 
like color and may show a laminated or fibrillated structure. The parasite is 
discussed in a later section. 

(7). Foreign Bodies. 

Examination of the sputum may reveal the presence of material which 
has lodged in the air-passages and been retained for long periods of time. 
Such bodies are coins, fish-bones, and cherry-stones. Heyfelder reports a case 
of the expectoration of a wooden cigar-holder 1 1 1/2 years after its disappearance. 

IV. Microscopic Examination. 

The microscopic examination of the sputum is almost the only one to 
which it is subjected at the present day. This is to be regretted, as much may 
be learned from a careful macroscopic examination. However, a microscopic 
examination reveals evidence which points to an absolute diagnosis more fre- 
quently than does the macroscopic examination. Before making the micro- 
scopic examination, it is wise to place the sputum in a flat-bottomed dish (Petri 
dish) which has half of its base blackened, so that the more suspicious particles 
may be selected for microscopic investigation. Some experience in this work 
is necessary, as one is frequently called upon to recognize material which is 
purely extraneous and has absolutely nothing to do with the sputum. Such 
material practically always comes from the buccal cavity and consists of frag- 
ments of various food-stuffs, such as bread, fruit pulp, meat fibers, vegetable 
tissue, and portions of tobacco leaf. Naturally, such material should not mis- 
lead one, but it very frequently does. The fragments of meat tissue contain 
elastic tissue fibers and may lead one to state that such material is present in the 
sputum, thus giving expression to the possibility of a diagnosis of incipient 
tuberculosis. 

A portion of the sputum selected for examination is taken up with a plati- 
num loop and spread in a thin layer upon a glass slide. It is then dried by 
passing the slide several times through the flame, care being taken not to burn 



IO 



DIAGNOSTIC METHODS. 



the specimen. The smear is allowed to cool and is then stained either with 
Loffler's methylene blue for general purposes or with special stains for the 
various specific organisms. 

(a). Pus-cells (Leucocytes). 

There is practically no specimen of sputum which does not contain leuco- 
cytes in larger or in smaller numbers. The true pus-cell is the polymorpho- 
nuclear neutrophile and appears in the sputum frequently filled with fat globules 
or pigment granules. In cases of asthma, the eosinophile cells are very abun- 
dant, while basophile cells may occasionally obtain. Although these cells 
are so frequent in asthma, a diagnosis should not rest upon such evidence alone, 









Fig. 2. — Objects found in the sputum {Latidois). i, Detritus and dust-particles; 2, pig- 
mented alveolar epithelium; 3, fatty degenerated and partially pigmented alveolar epithelium; 
4, alveolar epithelium showing myelin-degeneration; 5, free myelin forms; 6, 7, desquamated 
ciliated epithelium, partly changed and deprived of its cilia; 8, squamous epithelium from 
the mouth; 9, leucocytes; 10, elastic fibers; 11, fibrinous cast of a small bronchus; 12, lep- 
tothrix buccalis, together with cocci, bacilli, and spirochete; a, fatty acid crystals and free 
fatty granules; &, hematoidin; c, Charcot's crystals; d, cholesterin. 

as there seems to be a form of bronchitis, possibly of the tubercular variety, 
which has been named "eosinophilic bronchitis" from the large number of such 
cells observed. In pulmonary tuberculosis, which is not associated with a mixed 
infection, it is very common to find the small mononuclear leucocyte (lym- 
phocyte) in place of the polynuclear type. This finding is of such frequent 
occurrence that the writer is often led to search many slides for tubercle 
bacilli in case the organisms have not been found in the earlier specimens 
examined. Basophilic cells are not infrequently observed in the sputum. 
Pottenger 1 reports the presence of macro-phagocytes in a case of chronic hemop- 
tysis. The cytology of the sputum shows little of diagnostic value. 
1 Jour. Am. Med. Assn., 1912, LIX, 1794. 



THE SPUTUM. II 

The thin smears of sputum may be treated like blood-smears and stained 
with the same stains when one desires to study the cellular types present. 

(b). Red Blood-cells. 

These cells are frequently found in the sputum and may have much signifi- 
cance. They may occasionally arise from contamination with nasal or buccal 
discharges, but the true bronchial or pulmonary sputum usually shows them only 
in cases of hemorrhage or exudation. The rusty sputum of pneumonia contains 
large numbers of such cells and the hemorrhage from a tubercular cavity may 
be very extensive. These cells are occasionally well-preserved, but at times they 
are difficultly recognizable. They may be very much distorted in shape, so that 
their color and staining properties must be relied upon for differentiation. 

(c). Epithelial Cells. 

Various types of epithelial cells are found in the sputum. Pavement 
epithelium may come from the mouth, pharynx, and upper larynx. Cylindri- 
cal epithelium may be derived from the nose or the bronchi. These cylindrical 
cells may be ciliated, but rarely does one find these ciliated forms except in cases 
of asthma and bronchitis. Alveolar epithelial cells are present in normal sputa 
as they are constantly desquamated from all of the epithelial surfaces. These 
cells assume a large variety of forms and frequently show various types of de- 
generation. They are very numerous in bronchitis and in general inflammatory 
conditions of the lungs, but may occur in almost any condition, associated with 
irritation along the respiratory tract. These cells contain large numbers of 
granules which are probably referable to coal pigment. Such cells give the 
sputum a grayish or green color. An abundance of such cells in the sputum 
was designated in earlier times as "phthisis melanotica." Occasionally these 
alveolar cells are filled with fat globules. In other cases one finds the so-called 
myelin globules, whicn are irregular in shape, often showing concentric lines, 
with very little refractility, and of a dull greenish or blue appearance. The 
cell may be invisible and only the myelin appear as a large irregular mass. 
Much doubt exists as to the origin of these globules, but it is more probable 
that they represent simply the fatty products of degenerated protoplasm. 
These cells are especially frequent in the normal morning sputum, in acute or 
chronic influenza, and in the so-called desquamative catarrhal pneumonia, when 
they appear as small lumps resembling boiled sago. Occasionally free myelin 
globules are found in the sputum. These globules stain poorly with the aniline 
dyes, are stained yellow with iodin, but do not stain black with osmic acid or 
red with Sudan-Ill. 

Frequently these alveolar cells contain pigment granules, derivatives of 
hemoglobin. This material is in the form of amorphous granules of a brown 
color and seems to be identical with hemosiderin, but may become iron-free, 
when it more closely resembles hematoidin. Cells containing such granules are 
especially numerous in chronic heart disease and are styled, therefore, "heart- 
disease cells." They occur, however, in any condition in which red blood-cells 
escape into the alveoli and are found, therefore, in pneumonia, infarction of the 
lung, and hemorrhagic pulmonary tuberculosis. 



12 DIAGNOSTIC METHODS. 

(d). Elastic Tissue. 

The presence of elastic tissue in the sputum is indicative of destruction of 
the lung tissue and in many cases is found in the sputum before one can detect 
tubercle bacilli. It has, therefore, some importance in the early diagnosis of 
tuberculosis. When this elastic tissue is grouped in masses it is usually visible 
to the naked eye. However, one more frequently relies upon microscopic ex- 
amination for its detection. The method of Clark, as applied to the detection 
of elastic tissue, is usually the best one to follow. The sputum is placed upon 
a glass plate about 14 inches square and pressed out by a smaller one, about 6 
inches square, into a thin layer. The plates are then placed upon a dark back- 
ground and examined with a hand lens. Instead of these glass plates one may 
use Petri dishes. 

The elastic tissue fibers appear either in the form of distinct strands some- 
times grouped in an elongated network, in that of the alveolar type, in which 
the fibers preserve the outline of the alveoli and are long and branching, or in 
that from the arteries in which we may have a distinct sheet-like arrangement. 
These fibers are characterized by their undulating outline, their curling ends, 
their sharp edges and uniform diameter, their frequent branching, and their 
intense refractility. These characteristics are brought out both with the low 
and with the high power of the microscope. The fibrous tissue fibers are dif- 
ferentiated from elastic fibers by the fact that the former are present in bundles 
of fine wavy lines without the coarse black refractive appearance of elastic 
tissue. Chains of bacteria, especially the leptothrix forms, frequently interlace 
in such a way as to simulate the alveolar structure of elastic tissue. These 
chains differ, however, from elastic tissue in their refractility, in the absence 
of the wavy outline, and in their denser arrangement. The elastic tissue fibers 
derived from food substances have the same characteristics as the lung elastic 
fiber, with the exception that they are not arranged in the alveolar form and may 
at times be coarser and more irregular in outline than the pulmonary elastic tis- 
sue. Vegetable cells and fibers, as well as fatty acid crystals, which may occur in 
the sputum, should not mislead one into assuming the presence of elastic tissue. 

Should one wish to stain elastic tissue, he may use the orcein stain of Unna- 
Tanzer. This stain consists of one gram of orcein dissolved in a mixture of 80 
c.c. of 95 per cent, alcohol and 35 c.c. of distilled water, 40 drops of strong 
hydrochloric acid being added after solution is complete. In using this stain 
the elastic fibers are treated with a few c.c. of the dye and then warmed for 
five minutes, after which the preparation is decolorized with acid alcohol. 
The elastic tissue fibers will be stained a brownish violet by this process. 

If the elastic tissue is very small in amount we usually resort to chemical 
means for the isolation of this material. Ten c.c. of sputum are mixed with an 
equal volume of 10 per cent, sodium hydrate solution and the mixture is boiled 
until it becomes homogeneous. Four volumes of water are then added, the 
entire mass well mixed, and either allowed to stand or to settle by centrif ugation. 
In this way the constituents of the sputum are destroyed with the exception of 
the elastic tissue which has, however, become swollen and paler and does not 
have its characteristic appearance. 



THE SPUTUM. 13 

About 90 per cent, of cases showing elastic tissue in the sputum are of 
tuberculous origin, according to Dettweiler and Setzer. As the healing process 
begins and proceeds this elastic tissue gradually diminishes in amount, so that 
a constant presence or an increase in the amount indicates a progressive condi- 
tion. It is seen in abscess of the lungs, in bronchiectasis, in pulmonary infarct, 
and occasionally in pneumonia. It is found in cases of gangrene of the lungs, 
although there are many statements that it is digested by the trypsin-like fer- 
ment so common in such conditions. Osier states that he has never seen a case 
of gangrene of the lung in which elastic tissue fibre could not be found. 

(e). Crystals. 

Crystals are never found in the freshly formed sputum, being indicative 
of stagnation of the material within the body or of decomposition after being 
expectorated. 

(1). Fatty Acid Crystals. 

These crystals occur most frequently in the sputum of gangrene, putrid 
bronchitis and of chronic tuberculosis. They occur as distinct needles, either 
singly or in groups, may be short and relatively thick with pointed ends, or they 
may be long and very closely resemble elastic fiber. Pressure upon the cover- 
glass will produce varicosities in these crystals which do not appear in the case 
of elastic tissue. They are soluble in alkalies and in ether and melt into fat 
globules if the slide be warmed. 

(2). Cholesterin. 

Crystals of cholesterin are found in the sputum of chronic lung abscesses, 
empyema and chronic tuberculosis. They are not as frequent as are the fatty 
acid crystals, but are usually found associated with them when they are present. 
These crystals have a distinct rhomboid form with notched angles. 

(3). Hematoidin. 

Crystals of hematoidin occur very rarely in sputum, and then only 
when extravasation has taken place into the alveoli. They are found very rarely 
after direct hemorrhage unless the extravasated blood remains for some time 
in the alveoli. These crystals occur especially in abscess of the lungs, empyema, 
or perforating liver abscess. They are rhomboid or needle-shaped crystals, 
ruby-red in color, and may show small curved filaments projecting from the 
angles of the larger forms. 

(4). Leucin and Tyrosin. 

These substances are formed only by the decomposition of protein material 
and are found, therefore, in the putrid sputum of an empyema, or from a per- 
forating liver abscess, and in the very early discharges of true lung abscess. 
The tyrosin is found in the form of long refractive needles, frequently arranged 
in bundles, while the leucin appears as distinctly spherical masses with con- 
centric striations and radiating lines. 

(5). Magnesium-ammonium Phosphate. 

These crystals appear in the sputum under the same condition as do the 
preceding tyrosin and leucin crystals. They are usually the typical coffin-lid 



14 DIAGNOSTIC METHODS. 

crystals so common in decomposed urine, but may at times assume an irregular 
structure. 

(6). Calcium Oxalate. 

These crystals occur in the sputum in conditions associated with decom- 
position and appear either in the typical octahedral crystal with a cross con- 
necting the corners or as the more atypical dumb-bell shaped crystal. 

(7). Charcot-Leyden Crystals. 

These crystals are apparently derived from the eosinophile cells as they 
are more frequently present only in conditions in which the eosinophiles are 
very numerous, hence the term "leucocyte crystals." They increase in the 
sputum either from stagnation within the system or after being expectorated. 
They are associated in asthmatic attacks with the spirals of Curschmann, 
being frequently included in the meshes of the spiral. 

These crystals form straight, pointed, colorless, hexagonal, double pyra- 
mids, resembling a very much elongated diamond. They have sharp elon- 
gated points with clear-cut edges, are very brittle, are colorless, show little refrac- 
tility, and vary greatly in size. They may occur singly or in groups, forming 
either clusters or distinct Greek-cross types. They have been supposed to be 
identical with the spermin crystals of Bottcher, but the hexagonal type of the 
crystal as well as the facts that they do not show marked double refraction by 
polarized light and have but a single optical axis should serve to differentiate them 
from the spermin crystals. They are colored yellow with Florence's reagent 
and may be stained with the polychrome and other blood dyes. (See Semen.) 

(/). Bacteria. 
(r). Saprophytes. 

The bacteria found in the sputum are very numerous and under normal 
conditions are purely saprophytic. We may even at times find many truly 
pathogenic organisms in the sputum which are of no clinical significance, 
although one may be led into making a diagnosis without sufficient clinical 
evidence. 1 These saprophytes may occur in the fresh sputum or develop 
therein after the specimen has stood for some time. The various chromogenic 
bacteria are of particular interest as their development along the respiratory 
tract may so change the color of the sputum that the examiner may be led 
astray. 

Among the ordinary saprophytes found in the sputum we find representa- 
tives of the streptothrix 2 and the leptothrix groups. 3 Flexner and Warthin 
and Olney have reported the presence of a streptothrix, the streptothrix ep- 
pingeri, in the sputum of cases showing the clinical symptoms of pulmonary 
tuberculosis. These organisms are about four times as thick as the tubercle 
bacillus; when stained they are resistant to decolorization by acids, but are 
slowly decolorized by strong alcohol. Stained specimens are easily made with 

1 Boston (Interstate Med. Journ., 19 14, XXI, 330) reports the relatively frequent presence 
of tubercle bacilli in the sputum of acute colds, with disappearance of these organisms during 
convalescence. 

2 See Claypole, Jour. Exper. Med., 1912, XVII, 99. 

3 See Kato, Mitt. a. d. med. Fakultat der k. Univ., Tokyo, 1915, XIII, 441. 



THE SPUTUM. 



J 5 



the use of methylene-blue dyes or of Gram's method. The leptothrix group 
is particularly abundant in the mouth and is found in large numbers in the lungs 
in cases of pulmonary gangrene. 

Yeast fungi occur in the sputum at times, but rarely in the fresh specimens. 1 
They are oval or elliptical cells and are very refractive, sometimes resembling 




Fig. 3. — Aspergillus fumigatus. 



very closely fat droplets. Their appearance may vary from that of a simple 
oval cell without distinct limiting membrane to those with definite membrane 
and vacuoles. These cells are especially characterized by their tendency to 
throw out projections or buds at various points of their periphery. They vary 
in size from 1 to 40 microns in diameter. While these yeast fungi are usually 




Fig. 4. — Micrococcus catarrhalis. (From Emery s " Clinical Bacteriology. ") 

extraneous, cases are reported (Busse) in which pathogenic yeasts have been 
found in anomalous pulmonary conditions. These organisms stain with the 
ordinary aniline dyes and appear in some instances to be acid-fast (resisting 
decolorization by acid). They are gram-positive. 2 

1 See Breed, Arch. Int. Med., 1912, X, 108; Jour. Am. Med. Assn., 1913, LXI, 472. 

2 See Henrici, Jour. Med. Research, 19 14, XXX, 409. 



i6 



DIAGNOSTIC METHODS. 



Various types of molds are found in the sputum. Some of these appear 
to be distinctly pathogenic, while the majority are merely saprophytic. These 
molds are found in the true sputum only in cases associated with destructive 
processes of the lungs. There are at present many reports of cases showing that 
some of them at least may be distinctly answerable for primary infections. 

Among these pathogenic molds we find certain types of the Mucor, of 
the 130 varieties of which six are known to be distinctly pathogenic. Besides 




Fig. 5. — Budding forms of blastomycetes found in sputum. 
(From photograph by W. A. Pusey.) 



this type we find, as the most important pathogenic mold of the sputum the 
aspergillus fumigatus, 16 cases of pulmonary affection (pneumonomycosis 
aspergillina) having been traced by Sticker to this fungus. 1 Other types of the 
aspergillus are the flavus, niger, and the sub-fuscus. The penicillium glaucum 2 
and the Oidium albicans are occasionally found in the sputa. 

1 See von Teckman, Zur Lehre von der Pneumonomykosis aspergillina, Inaug. Dissert., 
Basel, 1 9 13. 

2 See Weidman, Proc. Path. Soc, Phila., 191 5, XVII, 62. 



THE SPUTUM. 17 

Among the bacteria which may be purely saprophytic we may find almost 
any of the pus-forming organisms. More frequently, however, when these or- 
ganisms are present in excessive numbers a contamination or a direct pathogenic 
influence should be suspected. A form known as the micrococcus tetragenus 
occurs both as a pathogenic and a harmless organism. It consists, as its name 
implies, of four cocci arranged in a square within a mucous capsule. It stains 
with the ordinary dyes and is Gram-positive. This organism is found, in its 
pathogenic state, in bronchitis, tubercular cavities, and hemorrhagic infarctions. ' 
The harmless form differs from the pathogenic type in the fact that it cannot 
be cultivated. 

The sarcinae are rarely found in the sputum. These organisms are 
somewhat smaller than those occurring in the stomach and are probably purely 
saprophytic in the sputum. They are found, however, in cases of putrid bron- 
chitis, especially when this occurs in emphysematous lungs, in gangrene, tuber- 
culosis, and pneumonia. 

The micrococcus catarrhalis is found frequently in the sputum as a sapro- 
phyte, but may become pathogenic especially in some epidemics of la grippe. 2 
It is larger than the ordinary staphylococcus, while its arrangement in lateral 
pairs and its Gram-negative properties obscure its differentiation from the 
gonococcus and meningococcus unless cultural methods be employed. (See 
last chapter.) 

In cases of general systemic blastomycosis, Eisendrath and Ormsby have 
found the blastomycetes in the sputum. They recommend the examination of 
the unstained specimens after the addition of 10 per cent, sodium hydrate solu- 
tion. In such preparations examined with a high-power dry lens, the typical re- 
fractile blastomycetes are observed. (See Parasitic Diseases.) 

(2). Pathogenic Types, 
(a). Tubercle Bacillus. 

This organism is the most important pathogenic type found in the sputum. 
Its detection is usually easy and should be attempted in all suspicious cases, as 
an early diagnosis may frequently save the life of the patient. In the days 
before the organism was recognizable, physicians based their diagnosis of con- 
sumption upon the macroscopic appearance of the sputum. While such ex- 
aminations frequently lead to a presumptive diagnosis of tuberculosis, nothing 
can settle the question except the microscopic examination of the sputum. 
This statement needs some modification in several ways. In the first place, 
specimens of tubercular sputum may not show the presence of the bacilli so that 
several examinations of sputum, collected at different periods, must be made. 
In the second place, the sputum may be examined in the very early stage of the 
disease and no tubercle bacilli be found, but in such cases the presence of elastic 
tissue fiber would be very significant of tubercular changes. In these days we 
have, fortunately, recourse to other diagnostic measures in case tubercle bacilli 
cannot be found in the sputum. I have reference here to the use of tuberculin, 

1 Steele (Jour. Am. Med. Assn., 1914, LXII, 930) reports a case of general septicemia due 
to this organism. 

2 Hastings and Niles, Jour. Exper. Med., 1911, XIII, 638. 



l8 DIAGNOSTIC METHODS. 

either introduced in the form of an injection as done by Koch, dropped into the 
eye as advocated by Calmette, as advised by Pirquet applied after the manner 
of vaccination, or used as an inunction as suggested by Moro. This test is 
usually certain and can be relied upon to settle the diagnosis. 

In examining the sputum microscopically the fine cheesy particles pre- 
viously mentioned are selected and smears of such material made upon glass 
slides. It is always advisable to make at least five such smears to insure 
definite results. It has frequently been found that many suspicious-looking 
sputa show no tubercle bacilli when subjected to the ordinary methods of 
examination. In order, therefore, to increase the possibility of positive results, 
the sputum should be rendered homogeneous and fluid. The best method of 
accomplishing this is, in the writer's opinion, Loeffler's 1 modification of the 
antiformin process of Uhlenhuth. The technic is as follows: 5, 10, or more 
c.c. of the sputum are placed in a flask and mixed with an equal quantity 
of a 50 per cent, solution of antiformin (a 10 per cent, solution of sodium hypo- 
chlorite containing 5 to 10 per cent, of sodium hydrate) and boiled for a period 
not exceeding 15 minutes. Solution occurs associated with considerable foam- 
ing and browning of the mixture. For every 10 c.c. of this solution are now 
added 1.5 c.c. of a mixture of 1 part of chloroform and 9 parts of alcohol. 
After thoroughly shaking to produce a fine emulsion, portions of the fluid are 
placed in sedimenting tubes, the tubes are corked, and centrifuged for fifteen 
minutes. The heavier elements collect in a film just above the chloroform, 
which film holds the tubercle bacilli owing to the marked affinity of chloro- 
form for the fatty and waxy material in these organisms. 2 The supernatant 
liquid is poured off and the film above mentioned is removed and placed upon 
a glass slide, the excess of fluid being taken up with filter-paper. As a fixa- 
tive a drop of egg-albumin, preserved with 1/2 per cent, carbolic acid, is added 
and a thin spread made by means of a second slide. This smear is allowed 
to dry and is then stained by one of following methods. This enrichment 
process of Lceffler furnishes preparations which often show a remarkable in- 
crease in numbers of tubercle bacilli as compared with those found by the 
usual smear methods. A further advantage of this method is that practically 
all organisms, with the exception of those of the acid-fast type to which the 
tubercle bacillus belongs, are destroyed. If the mixture be allowed to stand for 
a few hours instead of being boiled, one may obtain material for pure-culture or 
for inoculation purposes, as the secondary invaders are eliminated. For these 
latter purposes it is necessary to wash the sediment frequently with sterile water 
by means of centrifugation and decantation, in order to remove the excess of 
alkali present. This may, of course, diminish the number of tubercle bacilli 
and, at the same time, cause contamination of the specimen. In consequence, 
inoculation and cultural experiments with such material frequently give nega- 
tive results in known tubercular cases. 

A somewhat different method of eliminating the secondary invaders and of 

1 Deutsch. med. Wchnschr., 19 10, XXXVI, 1987. 

2 See Panzer, Ztschr. f. physiol. Chem., 1912, LXXVIII, 414; Wells, Interstate Med. Jour., 
1914, XXI, 221. 



THE SPUTUM. 19 

obtaining the tubercle bacillus in pure culture from the sputum (as well as other 
material) has been introduced by Petroff 1 and promises to become a very valuable 
aid in the study of the presence and characteristics of the tubercle bacillus. By 
the employment of a special culture medium to which gentian violet is added, 
Petroff takes advantage of the fact that this dye will inhibit the growth of many 
organisms but will not retard the development of the tubercle bacillus at the 
dilution at which it is used. Further, he adopts, as his homogenizing agent, an 
alkaline solution to which the tubercle bacillus is resistant while the viability of 
most other organisms is quite strongly affected. 

Preparation of the Culture Medium. 

Five hundred grams of beef or, preferably, veal are infused in 500 c.c. of 
a 15 per cent, solution of glycerin in water. Allow the infusion to stand for 24 
hours and squeeze the meat in a sterile press, collecting the juice in a sterile 
beaker. 

Sterilize the shells of one dozen fresh eggs by pouring hot water upon them. 
Carefully break the eggs into a sterile beaker and thoroughly mix the white and 
yolk with a sterile rod. Filter through sterile gauze into a sterile graduated 
cylinder. 

To two parts of the egg add one part of the meat infusion and add to this 
mixture sufficient 1 per cent, alcoholic solution of gentian violet to make a 
dilution of 1 to 10,000. Tube about 5 c.c. of this mixture in each sterile test- 
tube and inspissate the slanted tubes for three successive days: on the first day 
at 85 C. until all the medium is soldified; on the second and third days for not 
more than 1 hour at 75 C. (If this medium is to be used for the isolation of the 
bovine type of tubercle bacillus, the glycerin may be omitted in making the meat 
infusion.) 

Method. 

Equal parts of fresh sputum (about 5 c.c.) and 3 per cent, sodium hydrate 
solution are well shaken and placed in the incubator for 20 to 30 minutes until 
the sputum is fairly well digested. Neutralize this mixture to sterile litmus 
paper with normal hydrochloric or sulphuric acid. Centrifugalize at high speed 
for 10 minutes. Decant the supernatant fluid and spread the sediment over 
several slants of the culture medium. Incubate for 7 to 12 days. 

The cultural characteristics may be more or less variable, some of the colonies 
being of small pin-point size, while others are large and flat. Some of the types 
may decolorize the medium, while others appear violet. Morphologically, the 
organisms may vary from cocci to long rods. 

By this method of Petroff tubercle bacilli maybe detected in a comparatively 
short time in cases which have shown repeated negative results by the usual 
microscopic methods, as well as in cases which have not yielded positive results 
with animal inoculation following the antiformin method. It is simple, prac- 

1 Jour. Exper. Med., 1915, XXI, 38; Bull. Johns Hopkins Hosp., 1915, XXVI, 275; also 
Mitchell and Simmons, Jour. Am. Med. Assn., 191 5, LXV, 245. 



20 DIAGNOSTIC METHODS. 

tical and reliable. It is especially recommended as it tends to increase the 
possibility of diagnosis in obscure cases and may, even, supplant animal inocu- 
lation tests. 

Staining Characteristics. 

The methods of staining the tubercle bacillus depend upon the property, 
possessed by this organism, of taking up the aniline dyes with great difficulty, 
but, when once stained, of becoming just as resistant against decolorization. 
Breskman (Jour. Am. Med. Assn., 1910, LIV, 1591), DLxon (Ibid., 1913, LX, 
993) and, more recently, Wherry (Jour. Infect. Dis., 19 13, XIII. 144) have 
shown that the tubercle bacillus varies its acid-fast properties in its development 
on culture media, becoming more acid-fast or non-acid-fast according as the 
cultural conditions are or are not favorable for the synthesis of fat. Tamura 
(Ztschr. f. physiol. Chem., 19 13, LXXXVII, 85) demonstrates that this acid-fast 
property is due to the presence in the bacterial cell of an alcohol, mykol (C 29 H 56 - 
O), which is partially bound to some higher fatty acid in the form of an ester. 

Ziehl-Neelsen Method. 

The smears are made upon glass slides and are fixed by passing several 
times through a flame. The smear is covered with carbol-fuchsin solution *(a 
mixture of 90 parts of 5 per cent, carbolic acid water and 10 parts of a concen- 
trated alcoholic solution of fuchsin), and is then heated over a flame for one 
to three minutes in such a way that the staining solution steams, but does not 
boil. If the staining solution is heated too strongly the smear decolorizes less 
readily so that it is very good practice never to boil the staining solution. Where 
several slides are to be examined, the writer has found the copper plate of Ehrlich 
very useful. 2 The slides are laid upon the plate, are covered with the stain, and 
allowed to heat for 10 minutes. The usual technic is, however, to heat a 
single slide at one time, more of the stain being added as the first evaporates. 
Some workers find that the immersion of the smear in cold carbol-fuchsin for 
24 hours gives somewhat clearer pictures, but the time is too long for the ordi- 
nary laboratory diagnosis. Having thus stained the smear with the carbol- 
fuchsin solution, it is then decolorized. The tubercle bacillus is not only acid- 
fast, but also alcohol-fast, so that we use decolorizing agents containing both 
acid and alcohol. There are many acid-fast organisms known, such as the 
bacillus lepras, the smegma bacillus, the timothy bacillus, the butter bacillus 
and many saprophytic bacilli found in water, soil, and manure. 3 Few or none 
of these organisms are absolutely both alcohol and acid-fast, so that the use of 
the combined decolorizer will usually differentiate the tubercle bacillus. Many 
decolorizing agents have been advised, but the writer finds the use of a 10 per 
cent, solution of sulphuric acid in 95 per cent, alcohol very reliable. This 
decolorizing agent does not burn the specimen nor does it prevent the morpho- 
logical characteristics from appearing in a clear-cut way. Some workers ad- 
vise the use of 2 per cent, hydrochloric acid in 80 per cent, alcohol, while others 

1 See Verhoeff, Jour. Am. Med. Assn., 1912, LVHI, 1355. 

2 See Schmidt, Jour. Am. Med. Assn., 1915, LXIY, 823. 

3 Lycopodium spores, cork cells, honey-comb wax and other substances not infrequently 
found in the sputum are acid-fast and may prove very misleading. 



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THE SPUTUM. 21 

use 25 per cent, nitric acid, followed by alcohol. The technic of decolori- 
zation is as follows: Wash the smear, which has been stained with carbol- 
fuchsin, in water and flood the specimen with the decolorizing solution until 
only the faintest pink color is seen in the smear. It frequently happens that 
the thicker portions of the smear resist this decolorizing so that it may be neces- 
sary either to make a new smear or to examine merely the portions which have 
been decolorized. After decolorization is complete the specimen is washed 
in water and counter-stained with Loffler's methylene blue (saturated alcoholic 
solution of methylene blue 30 c.c, 100 c.c. of a 1 to 10,000 aqueous potassium 
hydrate solution) for a few seconds, after which the specimen is washed with 
water, dried between filter-paper, and examined with the oil immersion lens. 
In such preparations the tubercle bacilli are seen as bright red rods, some- 
what bent, sometimes much curved and occasionally showing distinct branching 
forms. l In a few preparations one may find the curves of the bacillus so marked 
that a very close resemblance to the spirillar forms obtains. These organisms 
occasionally show a distinct beading, giving the appearance of bright red cocci. 2 
The size of these organisms varies from 1 1/2 to 3 1/2 microns and about 2/10 
microns in width. They may be single or arranged in clumps, sometimes in 
the form of distinct crosses, sometimes parallel, and very frequently forming 
acute angles by the joining of two bacilli. 

Gabbet's Method. 

This method is much more simple than the preceding, but is not as reliable. 
By this method the decolorization and counter-staining are carried out in one 
operation. The smears are prepared as previously described and stained with 
the carbol-fuchsin solution. The excess of the staining solution is drained off 
without washing and is replaced by Gabbet's methylene blue solution (methy- 
lene blue 2 grams, sulphuric acid 25 c.c, water 75 c.c). This solution is allowed 
to act for one to three minutes and is then washed off with water and the 
specimen dried and examined. The tubercle bacilli will appear as bright red 
rods as previously described, while the other organisms as well as the various 
cellular types will be stained blue. 

This method is not as reliable as is the former, owing to the fact that the 
alcohol-fast bacilli resist decolorization and may confuse one in making the 
diagnosis. Moreover, the use of the strong acid may cause decolorization of 
some of the tubercle bacilli and will, therefore, give rise to wrong ideas. These 
methods are the ones usually followed in routine laboratory work for the de- 
tection of the tubercle bacillus. 

Pappenheim's Method. 

The technic of this method is as follows: The preliminary staining 
is carried out with carbol-fuchsin solution as previously outlined. The speci- 
men is then drained and covered with the decolorizing solution, which is made 
by dissolving 1 gram of rosolic acid in 100 c.c. of absolute alcohol, saturating 
the mixture with methylene blue and adding 20 parts of glycerin. This solution 

1 See Dixon, Jour. Am. Med. Assn., 1913, LX, 993 and 1294. 

2 See Babes, Berl. klin. Wchnschr., 1914, LI, 501. 



22 DIAGNOSTIC METHODS. 

is drained off slowly and the process repeated several times. The slide is then 
washed in water, dried between blotting-paper and examined with the immersion 
lens. The tubercle bacilli are stained red and the other organisms blue. 

Much's Method. 

As is well known, certain specimens of undoubted tubercular sputum as 
well as tissues frequently do not show tubercle bacilli when any of the above 
methods of staining are used, owing to the facts that neither is every acid- 
alcohol-fast bacillus the tubercle bacillus nor is every tubercle bacillus abso- 
lutely acid-fast. It is to Spengler 1 and, more especially, to Much 2 that we 
are indebted for a method which will demonstrate these non-acid-fast types of 
tubercle bacilli, which are distinctly granular, frequently appearing as mere 
granules rather than as true bacilli. These granules, under certain unknown 
conditions, change into true bacillary types and vice versa. 3 Much's method is 
a modified Gram method, the tubercle bacilli being Gram-positive. 4 Prepare 
smears as described above. Cover this smear with a carbol-methyl violet 
solution (10 c.c. of a saturated alcoholic solution of Gruebler's methyl violet 
B. N. mixed with 90 c.c. of 2 per cent, aqueous carbolic acid solution) and 
heat to boiling several times. Wash off stain with water and cover smear with 
Lugol's solution (iodine 1 gram, potassium iodide 2 grams, water 300 c.c.) for 
5 minutes. Wash with water and treat with 5 per cent, nitric acid for one 
minute and follow this with 3 per cent, hydrochloric acid for 10 seconds. 
Without washing place the slide in a mixture of equal parts of acetone and 
absolute alcohol until the smear is colorless. Wash with distilled water and 
counter-stain with 1 per cent, aqueous solution of saf ranin for a few seconds. Wash 
in water, dry thoroughly, and examine with the immersion lens. The tubercle 
bacilli and the granular forms appear bluish while the other organisms are red. 5 

Value of Examinations. 

Brown 6 in an able manner has summed up the value of the sputum examina- 
tion for tubercle bacilli. He gives as his reasons for believing that one should 
be guarded in forming an opinion of the prognosis of certain cases the follow- 
ing points: (1) Many of the tubercle bacilli may not be stained at all. (2) Old 
foci may give off very few and young foci no bacilli at all. (3) By the occlu- 
sion of a bronchus the contents of a focus may be shut off entirely for a time 
and thus the expelled sputum may contain a large number of tubercle bacilli. 
(4) The organisms may be present one day and not again for months. (5) 
The organisms may be abundant in one part of a specimen and none be found 
in others. (6) Some patients with fatal tuberculosis (caseous pneumonia or 
acute miliary tuberculosis) may have no bacilli in the sputum, while in other 
cases the organisms are present even before physical signs obtain. (7) In 

1 Deutsch. med. Wchnschr., 1905, XXXI ; 1228 and 1353; Ibid., 1907, XXXIII, 337. 

2 Beitr. z. Klin. d. Tuberk., 1907, VIII, 85 and 357. 

3 See Korber, Deutsch. med. Wchnschr., 1912, XXXVIII, 1494; Weiner, Munch, med. 
Wchnschr., 1914, LI, 1838; Bruckner, Ztschr. f. klin. Med., 1914, LXXX, 360; Mircoli, Gazz. 
d. osp., 1914, XXXV, 617; Tallo, Gazz. internaz. di med., 1915, XVIII, 241. 

4 See Benians, Jour. Path, and Bacterid., 1912, XVII, 199. 

5 See Mas y Magro, Rev. valenc. de cien. med., 1913, XV, 37. 

6 Montreal Med. Jour., 1901, XXX, 769; Jour. Am. Med. Assn., 1903, XL, 514. 



THE SPUTUM. 23 

severe cases with bronchitis the secretion of the bronchus will dilute the sputum 
and give the appearance of a reduction in the number of organisms. 

While the number of bacilli in the sputum may thus vary, it is usually in 
direct ratio to the severity of the disease, although for the reasons above men- 
tioned too much reliance should not be placed upon the number of organisms 
found. Brown recommends the use of a somewhat modified Gaffky's table 
in judging of the prognosis in any particular case. The cases are classified as 
follows, being designated by the Roman numerals: 

I. Only one to four bacilli in whole preparation. 
II. Only one on an average in many fields. 

III. Only one on an average in each field. 

IV. Two to three on an average in each field. 
V. Four to six on an average in each field. 

VI. Seven to twelve on an average in each field. 

VII. Thirteen to twenty-five on an average in each field. 

VIII. About fifty on an average in each field. 

IX. About 100 on an average in each field. 

There has been some attempt to base a prognosis upon the form and group- 
ing of the tubercle bacilli, the short rods indicating a rapid growth while the 
longer form shows a slower development. 1 The continued expectoration of 
large numbers of bacilli would indicate a cavity, while the sudden increase in 
numbers associated with an increase in the cellular elements would point to 
lung disintegration. A steady decrease over a long period of time would indi- 
cate improvement. It should be stated as a working rule that the finding of a 
single or a very few organisms in the sputum should be looked upon with sus- 
picion, but that an absolute diagnosis should be made only after repeated ex- 
amination has shown the presence of the tubercle bacilli. 2 The worker will 
find in the study of preparations stained as above that artefacts are very common 
so that a hurried examination is never warranted. 

The sputum in tubercular cases rarely shows tubercle bacilli in pure cul- 
ture. One usually finds large numbers of streptococci, staphylococci, micro- 
cocci catarrhalis, and frequently influenza bacilli and pneumococci. Pus cells 
may be few or many. Blood-cells may or may not be present, while elastic tissue 
fiber is very frequent, appearing in many cases before tubercle bacilli can be 
demonstrated. It should be remembered that mixed infections may be so 
marked that few, if any, tubercle bacilli appear in the sputum. The writer has 
frequently found sputa showing fairly large numbers of tubercle bacilli becoming 
practically negative for these organisms if the sputum be allowed to stand 
exposed to the air for 48 hours. The explanation is that the other organisms so 
far outgrow the tubercle bacillus that they prevent any further development of 
this latter organism and bring about such degeneration that the staining qualities 

1 See Wilson, Lancet, 1914, II, 1198; Cohn, Beitr. z. Klin. d. Tuberk., 1914, XXXI, 1; 
Kirchenstein, Ibid., 33. 

2 The type of tubercle bacillus found in the sputum is practically always the human strain. 
Only seven cases are reported in the literature of infection with the bovine type (see Griffith, 
Brit. Med. Jour., 1914, 1, 1171; Mollers, Deutsch. Med. Wchnschr., 1914, XL, 1299). 



24 DIAGNOSTIC METHODS. 

of the tubercle bacillus are markedly affected. This fact has been taken ad- 
vantage of in the clinical use of injections of pus organisms into tubercular joints. 

(b). Lepra Bacillus. 

The bacillus leprae, first described by Hansen, is a small slender bacillus 
from 4 to 6 microns in length and surrounded by a slimy envelope. These 
bacilli behave toward staining reagents very much like the tubercle bacillus, 
but are less resistant toward acid and alcohol than is the tubercle bacillus, so that 
a differentiation is possible provided decolorization is rather severe. The stained 
bacilli often show clear spots or appear as if made up of stained granules. 

These organisms may be found in many cases of leprosy in the sputum 
or nasal secretion, so that in doubtful cases a differentiation is necessary. 
While these organisms stain much more easily than do the tubercle bacilli and 
are more easily decolorized, it may be necessary to resort to inoculation ex- 
periments to make the absolute differentiation. 1 

(c). Smegma Bacillus. 

This bacillus may be found normally in the saliva, coating of the tongue, 
the tartar of the teeth, and in the crypts of the tonsils. Pathologically, it may 
be found in cases of simple bronchitis, in the sputum in cases of gangrene of the 
lungs, and in the suppurative discharges from the ears. While these organisms 
are much more commonly confused with the tubercle bacillus when the urine 
is examined than when the sputum is investigated, yet they must be borne in 
mind in every sputum examination. It may be necessary to resort to inoculation 
experiments to decide the question, but ordinarily the use of the differentiating 
stain of Pappenheim (previously described) as well as the following method 
of Bunge and Trantenroth may be used. After fixation of the smear, the fat is 
removed by soaking the specimen in absolute alcohol. The preparation is now 
covered with a 5 per cent, solution of chromic acid for 15 minutes, after which it is 
washed with water. The smear is stained with carbol-fuchsin, decolorized 
with 16 per cent, sulphuric acid for three minutes, and is then counter-stained 
for five minutes in a concentrated alcoholic solution of methylene blue. This 
method shows the tubercle bacillus as distinct red, while the smegma bacillus 
is blue. Wliile both of these methods of differentiation are usually applicable, 
yet one occasionally finds the smegma bacillus resisting the action of 16 per 
cent, sulphuric acid for 30 minutes and occasionally of strong alcohol for 12 
hours. 

(d). The Timothy Bacillus. 

This organism is present in the mouth reaching it through the medium 
of butter and milk, which may contain it in large numbers. These bacilli 
resist the decolorizing action of both alcohol and acid to almost the same extent as 
do the tubercle bacilli, but they usually appear as somewhat longer and thicker 

1 See Duval, Jour. Exper. Med., 1911, XIII, 365; also Duval and Wellman, Jour. Am. 
Med. Assn., 1912, LVIII, 1427; Clegg cultivates this bacillus in symbiosis with amoebasand 
cholera vibrios (Philip. Jour. Sc, Sec. B., 1909, IV, 403) ; Smith, Lynch and Rivas have demon- 
strated the transmissibility of the lepra bacillus by the bed-bug (Am. Jour. Med. Sc, 1913, 
CXLVI, 671). See, also, Wolbach and Honeij, Jour. Med. Research, 1913, XXIX, 367; 
Honeij and Parker, Ibid., 19 14, XXX, 127. 



THE SPUTUM. 



2 5 



rods. Strangely enough, this organism produces a lesion in guinea-pigs which re- 
sembles very closely that of true tuberculosis, so that the inoculation test will 
not always be conclusive unless other animals are inoculated with material from 
the first one, in which case no lesions develop in the later animals. Fortunately, 



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Fig. 6. — Diplococcus pneumoniae. (Williams.) 

the cultural peculiarities of this organism are markedly different from those 
of the tubercle bacillus, as the former develops readily on the ordinary culture 
media. 

(e). The Pneumococcus (Diplococcus pneumoniae). 

This organism, discovered by Frankel and elaborated by Weichselbaum, 
is generally recognized as the etiologic factor in cases of acute croupous pneu- 
monia, although other organisms not infrequently give 
rise to this condition. It is found in large numbers in 
the sputum and other exudates, appearing as a small 
slightly elongated conical or lance-shaped coccus, which 
shows a marked tendency to occur in pairs (diplococci) 
with the broader ends in apposition. Occasionally it is 
arranged in short chains resembling streptococci. 1 In 
exudates and in the blood this organism is usually en- 
veloped by a well-defined hyaline zone or capsule. In 
clinical work it is rarely necessary to stain this capsule 
to identify the organism but if this be desirable it is best done by the method 
of Rosenow. 2 It stains well with the ordinary dyes and is Gram-positive. 

Friedlander's Bacillus (bacillus mucosus capsulatus) is occasionally found 
in some cases of lobar and lobular pneumonia and occasionally may be con- 

1 See Dochez and Avery (Jour. Exper. Med., 1915, XXI, 114; Ibid., XXII, 105) and Lyall 
(Ibid., 1915, XXI, 146) for studies of the types of pneumococci occurring in normal and patho- 
logical throats. 

2 Jour. Infect. Dis., 1911, IX, 1. 




Fig. 7. — Friedlander's 
bacillus (above): pneu- 
mococcus (below). 
(Greene.) 



26 DIAGNOSTIC METHODS. 

sidered the etiologic factor in such conditions, although it is usually a 
secondary invader. These bacilli grow readily on artificial media, are en- 
capsulated and stain easily with the ordinary dyes but are Gram-negative. 

(/). The Influenza Bacillus. 

This bacillus, known as Pfeiffer's bacillus, is found in the bronchial 
sputum, especially in the pulmonary type of this disease. 1 The most char- 
acteristic sputum is greenish-yellow in color with lumps of pus in num- 
mular form. The organisms are found in such sputum as small, short 
bacilli measuring 2/10 to 3/10 micron in breadth by 5/10 micron in length. 
They usually occur singly, but may form chains. In the stained specimens 
these organisms show distinct polar stainings, appearing frequently as diplo- 
cocci. They are stained with dilute carbol-fuchsin solution, faintly with the 

ordinary methylene blue solution, or 
are identified by their Gram-negative 
characteristics. The best counter- 
stain used in the Gram method is 
either Bismarck brown or safranin, 
the organisms appearing both intra- 
and extra-cellular. 

(g). The Bacillus Pertussis. 

This organism, discovered by 
Bordet and Gengou, and elaborated 
by Klimenko, has been frequently 
found in the sputum in cases of 
whooping-cough. It resembles very 
Fig. 8.— Bacillus influenzae in sputum. closely the influenza bacillus, appear- 

ed bbott.) J . . . J , .„. 

ing as short, plump, ovoid bacilli, 

with rounded ends and lying singly or in small groups between the pus and 
epithelial cells. It stains feebly with the usual dyes often showing bipolar 
staining and is Gram-negative. This organism is rarely intra-cellular and 
may thus be distinguished from the influenza bacillus. 

(h). Typhoid Bacillus. 

This organism has been found in the sputum in typhoid fever cases show- 
ing a coexistent bronchitis or pneumonia. The sputum is usually hemorrhagic 
in character and shows the bacilli as short 5 thick rods, staining with the ordinary 
dyes and negative to Gram's stain. 

(i). Staphylococcus and Streptococcus Pyogenes. 

These organisms are found in practically every sputum examined and can 
be identified only by the use of cultural methods. They stain well with any 

1 See Davis (Jour. Am. Med. Assn., 1915, LXIV, 1814) for a discussion of the role of this 
organism, as well as other members of the hemoglobinophilic group as secondary invaders in 
respiratory infections. Also, Wollstein, Jour. Exper. Med., 1915, XXII, 445. 




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THE SPUTUM. 27 

of the aniline dyes and are Gram-positive. Their presence in the sputum has 
little pathologic significance. 1 

(/). The Bacillus Pestis. 

This bacillus of bubonic plague was discovered by Kitasato and Yersin in 
1894. It is a short, thick bacillus, measuring from 0.8 to 2 microns in length and 
from 0.4 to 0.8 micron in thickness. A capsule may be usually made out and 
the stained organism frequently resembles a diplococcus, owing to the intense 
polar staining with intermediate faint staining. These organisms are Gram- 
negative. 

The bacillus pestis is found in the sputum of persons suffering from the 
pneumonic type of this disease and should be recognized owing to the markedly 
infective character of the material. 2 It may be necessary absolutely to identify 
the organism by inoculation and cultural experiments. 

(k). The Bacillus Anthracis. 

The sputum of cases of pulmonary anthrax may contain large numbers 
of these bacilli. These organisms are from 5 to 10 microns in length and from 
1 to 1 1/2 in breadth. They are frequently grouped in long segmented threads, 
the segments varying in length, but usually being two or three times as long as 
broad. Occasionally these bacilli may be single, but are usually multiple. 
They form oval spores in the middle of the short segments. The organism 
stains with the ordinary dyes and is also Gram-positive. For absolute identifica- 
tion cultural and inoculation experiments, the latter into white mice, may 
be necessary, but the morphological characteristics will usually identify it. 

(/). The Bacillus Mallei. 

This organism of glanders is found in the sputum in the pulmonary form 
of this disease. Morphologically, there is nothing characteristic in the ap- 
pearance of this organism beyond the fact of the presence of faintly staining 
areas in the protoplasm of the rather long bacilli. These organisms stain by 
Gram's method as well as with the ordinary aniline dyes. For a final diagno- 
sis inoculation into a guinea-pig should be made. 

(m). Actinomyces Hominis (Ray Fungus). 

This fungus, which gives rise to the condition known as lumpy jaw in 
cattle, occasionally infects man, causing pulmonary conditions designated strep- 
tothricosis. The mucopurulent sputum in such conditions contains elastic tissue 
and small sulphur-yellow granules which are visible to the naked eye and are 
the characteristic findings of such cases. Macroscopically these granules are 
yellowish, grayish, greenish, or brownish in color, and are sometimes abundant 
and sometimes scarce. 3 They are very friable, and when gently crushed beneath 

1 The streptococcus viridans is not infrequently found in association with infections of the 
upper respiratory tract. See Cecil, Arch. Int. Med., 1915, XV, 150. Luetscher (Ibid., 1915, 
XVI, 657) discusses the importance of cultural methods in non-tubercular infections. 

2 See Strong and Teague, Philip. Jour. Trop. Med., 1012, VII, 187. 

3 Davis (Jour. Infect. Dis., 1914, XIV, 144) calls attention to the actinomyces-like granules 
found in the crypts of tonsils and composed of bacilli, streptococci and spirilla. See, also, 
Rullmann, Mtinch. med. Wchnschr., 1914, LXI, 1899; Claypole, Arch. Int. Med., 1914, XIV, 
104; Davis, Ibid., I; Cope, Brit. Jour. Surg., 1915, III, 55- 



28 



DIAGNOSTIC METHODS. 



the cover-glass and examined microscopically appear to have broken up into 
hyaline rounded masses at the margins of which, on close inspection, fine radial 
striations or filaments or hyaline club-shaped bodies, all closely set together, 
may be seen. The club-shaped bodies are variable in size and are composed of 
a hyaline refringent substance. In the granules obtained from the lesions in 
man the club-shaped formations are much ]ess frequently observed than those 
obtained from the lesions in cattle (Mallory and Wright). If cover-glass 
preparations be made and stained with Gram's method, one will usually find 
isolated and matted filaments, many of which may be seen to branch, in addition 




Fig. 9. — Actinomyces. (Williams.) 

to longer and shorter fragments of filaments and fine detritus of the same. If 
clubs are present in the granules they may be found scattered throughout the 
preparations. In this connection see Bridge. 1 

(g). Animal Parasites. 
(a). Amebae. 
Artault has described a unicellular ameboid body which resembles very 
closely a leucocyte when stained, but, while motile, differs in refractility and 
staining quality. This he calls the ameba pulmonalis. In cases of perforating 
liver abscess the true amebae coli may be found in the sputum, and, according 
to Flexner, in cases of abscess of the jaw communicating with the mouth. 2 These 
amebae will be discussed fully in the chapter on Feces, to which the reader is 
referred. It should be noted here that these organisms may not be numerous 
so that many slides may have to be examined. 

(b). Flagellates. 
Flagellated organisms, such as the trichomonas pulmonalis and the 
cercomonads, are found in the sputum associated with Dittrich's plugs incases 
of gangrene, putrid bronchitis, and tubercular cavity formation. The tricho- 
monas is probably identical with the trichomonas vaginalis or intestinalis. 

1 Jour. Am. Med. Assn., 1911, LVII, 1501; also, Schlegel, Kolle and Wassermann's 
Handb. d. path. Mikroorg., 1912, V, 301. 

2 These are probably identical with the endameba? gingivalis found in the mouth in cases of 
pyorrhea. (See next chapter.) 



THE SPUTUM. 



2 9 



(c). Cestodes. 
Not infrequently the lung is the seat of infection with the taenia echinococcus. x 
In such cases various foreign bodies, such as fragments of membranes, scolices, 
hooklets, and cysts, may be found in the sputum. Such formations may, also, 
be found in cases of liver abscess perforating into the lung. Any one of the 
above formations is characteristic of this condition. The parasite will be dis- 
cussed fully in the section on Feces, to which the reader is referred. 

The sputum in such cases is usually purulent or mucopurulent and may 
be tinged with blood. This sputum may be expectorated over a long period 




Fig. 10. — Paragonimus westermanii; (ventral view); 10X1. A, oral sucker; B, ceca; 
D, acetabulum; E, genital pore; F, uterus; G, ovary; H, testicles; 7, vitelline glands; 
K, excretory canal; L, excretory pore. (Tyson after Braun.) 

of time and may even contain tubercle bacilli from a coexistent tuberculosis. 
A distinguishing point between the sputum of pulmonary echinococcus disease 
and that of a perforating liver abscess is that the sputum in the latter is usually 
bile stained. 2 



{d). Trematodes. 

The most common of this class of animal parasites is the ordinary "lung 

fluke," which has been called also distoma pulmonale, distoma Westermanii, 

distoma Ringeri, and Paragonimus Westermanii. The eggs of this parasite 

are much more frequently found in the sputum than are the parasites them- 

1 See Filia, Pensiero med., 1914, IV, 741; also, Ortali, Gazz. d. osp., 1915, XXXVI, 225. 

2 See Maliwa, Munch, med. Wchnschr., 1914, LXI, 2367. 



3° 



DIAGNOSTIC METHODS. 



selves, so that the diagnosis will rest with the finding of these ova. These 
eggs measure from 80 to 100 microns in length and 40 to 60 microns in 
width. They are brownish in color, oval in shape, have a smooth thin 
shell and a lid near one end which is quite characteristic The parasite is 
from 8 to 10 mm. long, 4 to 6 mm. wide, and is very markedly rounded 
anteriorly, being nearly as thick as broad. 

The sputum in such cases is usually small in 
amount, is very tenacious, and is reddish or rusty due 
to admixture of blood with the mucus. Frequently no 
blood is found, in which case the sputum will still be of 
a yellowish or brown color due to the eggs themselves. 
The sputum, also, contains many spirals, which resemble 
very closely the Curschmann spiral and, also, the 
Charcot-Leyden crystals. 

The eggs of another species of distoma, the dis- 
toma hematobium, have been found in the sputum by 
Manson. Gage 1 has reported the finding of the larvae 
of strongyloides intestinalis in the sputum. 




Fig. 11. — Ovum of 
paragonimus wester- 
manii, from sputum: 
1000 X 1. {Tyson after 
Braun.) 



V. The Sputa in Disease. 



(1). Pulmonary Tuberculosis. 

It has been truly said by Brown that pulmonary tuberculosis has no charac- 
teristic form of sputum The amount voided may vary from the very slight type 
of fibroid tuberculosis to the very abundant sputum of cavity formation. It 
is to be said that the amount of pus will usually depend upon the extent of the 
secondary infection, although caseous degeneration may lead to the expectora- 
tion of large amounts of material resembling pus. 

In the early cases of pulmonary tuberculosis we may find a small amount 
of sputum which is expectorated only in the morning. This may be very 
tenacious and resemble very much the sago-like sputum previously mentioned. 
Sooner or later depending upon the extension of the disease, there will appear 
small caseous particles which are very suggestive. As ulcerative processes 
proceed, the sputum becomes more profuse, yellowish or greenish in color, and 
muco-purulent in character. In any stage of this ulcerative tuberculous con- 
dition we may find blood in amounts ranging from a few blood-cells to a sputum 
loaded with blood from a hemorrhagic focus. Likewise we will find elastic 
tissue in more or less amount and tubercle bacilli varying from a few to many in 
each field. The color of the tubercular sputum may range through all the 
shades of the spectrum, the greenish shade being associated with a most marked 
decomposition. As stated previously, the most suspicious looking sputa fre- 
quently contain no tubercle bacilli, so that frequent search must be made for 
these organisms which are the only diagnostic point of the sputum in this 
condition. 



1 Arch. Int. Med., ion, VII, 561. 



THE SPUTUM. 3 1 

(2). Croupous Pneumonia. 

The early sputum of acute lobar pneumonia is usually yellowish-red in 
color and very tenacious in consistency. In some cases the sputum is mucoid 
and abundant for a few days, but soon takes on the characteristic reddish color 
from the presence of unchanged red blood-cells. Its consistency is so great that 
the receptacle may be inverted without allowing any material to run out. The 
characteristic rusty sputum, which is found when the exudation into the alveoli 
is taking place, is homogeneous, glairy, very tenacious, and deep red in color. 
This rusty sputum, while characteristic of pneumonia, is sometimes replaced 
by one ranging in color from a yellow to a green. These colors are due to dif- 
ferent oxidation products of hemoglobin, and are, perhaps, more frequently 
observed in the stage of resolution when the sputum becomes less tenacious and 
more abundant. The greenish sputa in pneumonic conditions have some im- 
portance. This coloration may be due to a coincident jaundice or may arise 
from delayed resolution, especially when the exudate has been particularly 
hemorrhagic. It is, moreover, sometimes an indication that a true tubercular 
condition has intervened and, hence, that the prognosis must be guarded. 

The so-called prune-juice sputum usually indicates a severe type of the 
disease, while at times it may signify merely a beginning resolution. 1 

Fibrinous coagula are found, according to Osier, in every case in which 
search is made. These may vary from very small bronchial casts to very large 
branching types. Curschmann's spirals as well as the Charcot-Leyden crystals 
are frequently observed. The characteristic organism of this condition, the 
diplococcus lanceolatus of Frankel, is usually found, but has only incidental im- 
portance, as it is so frequently present in the sputum of normal individuals. 2 

(3). Bronchopneumonia. 

The sputum of this disease is rarely characteristic. It partakes of both 
the type of a bronchitic and a pneumonic sputum. It may, therefore, contain 
much mucus and pus, may be viscid, may be streaked with blood, but is rarely 
so distinctly rusty as in the croupous type of this disease. As the disease is so 
limited in extent it is more or less rare to find an abundant sputum or to observe 
fibrinous coagula. Microscopical examination shows various organisms, but 
nothing diagnostic. 

(4). Acute Bronchitis. 

The sputum in this condition is very scanty in the early stages, is usually 
very tenacious and is expelled with difficulty. This early bronchitic sputum 
is known as "sputum crudum" and consists of practically pure mucin, containing 
within its meshes a few leucocytes, red cells, bronchial epithelial cells, and a few 
myelin drops. 

After a few days the sputum is increased in amount, becomes less viscid, 

Pacini (Interstate Med. Jour., 1912, XIX, 536) has advanced the following re- 
action as characteristic of early pneumonic sputum: Mix 1 volume of sputum with 10 vol- 
umes of distilled water, agitate for 5 minutes and filter. To a test-tube containing 10 c.c. of 
distilled water add 5 drops of a 1 per cent, aqueous methyl- violet solution. Add to this latter 
10 drops of the sputum filtrate. A positive reaction is shown by the appearance of a distinct 
red color. 

2 See Rosenberger and Dorworth, New York Med. Jour., 1Q13, XCVII, 532. 



32 DIAGNOSTIC METHODS. 

and assumes the type of a distinct mucopurulent sputum. This sputum, 
called the sputum coctum, contains numerous pus-cells, is yellow or yellowish- 
green in color, shows the presence of large numbers of red cells, as a rule, and 
an increase of the polynuclear leucocytes over the mononuclear form. These 
mononuclear forms are more characteristic of the sputum of true tubercular 
conditions. Fat may be found, either in isolated drops or in large masses. As 
improvement in the condition occurs, the sputum becomes more abundant, 
and more distinctly purulent, and then gradually diminishes until it ceases. 
The sputum of acute bronchitis may give much information as to the course of 
the disease, as the transition from the viscid mucoid sputum through the abun- 
dant purulent stage to the final cessation is quite characteristic. 

(5). Chronic Bronchitis. 

(a). Simple Chronic Bronchitis. 

In most of these cases the sputum is either very little in amount or is much 
more abundant than in the acute forms. Such cases of simple chronic bron- 
chitis are usually those following the acute type of the disease in which we find 
the expectoration, for long periods of time, of a tenacious, viscid, and scanty 
sputum. Later it may become more abundant and muco-purulent, and may 
have a dark color and a distinctly foul odor. 

In the type of chronic bronchitis associated with cardiac disease we find 
large amounts of blood which may be fresh or changed, giving the typical 
prune-juice appearance. In such sputum we frequently find large numbers 
of the so-called "heart-disease cell" which have been previously described. 

(b). Putrid Bronchitis. 

This condition is brought about by dilatation of the bronchial tubes fol- 
lowing a chronic bronchitis. The sputum lies stagnant in these dilated bronchi 
so that it decomposes to a great extent. The sputum in such cases is very 
abundant, is of an ash-gray or brown color, is markedly purulent, and has a very 
disagreeable odor. On standing it separates into the three layers which have 
been previously discussed. In such conditions no elastic tissue fiber is found, 
so that we have here a differentiation from gangrenous or tuberculous pul- 
monary conditions. The sputum in this condition is very similar to that found 
in bronchiectasis, which is usually associated with decomposition of the sputum. 
Whether a diagnosis is possible between a straight putrid bronchitis and bron- 
chiectasis is doubtful, if one relies merely upon the sputum. The sputum in 
bronchiectasis occurs usually in the morning and is then very profuse. It 
shows, however, the characteristics of the sputum of putrid bronchitis, but is 
more commonly associated with the presence of pus, while 50 per cent, of cases 
show more or less profuse hemorrhage. 

(c). Fibrinous Bronchitis. 

The chief characteristic of this condition is the expectoration of more or 
less perfect bronchial casts. These may be single or may be distinctly branch- 
ing, showing the arrangement of the entire bronchial tree. This condition 
occurs quite frequently associated with many febrile diseases, but in the dis- 



THE SPUTUM. 



33 



cussion at this point we have reference to the idiopathic type of the disease. 
The sputum in this latter class of diseases is mucoid and very abundant in the 
earlier stages. After a few days there is expectorated, following a severe 
coughing spell, a bronchial cast. This expectoration is usually tinged with 
blood. Such casts may be expectorated over long periods of time and their 
form may vary as previously described. 

(6). Bronchial asthma. 

The sputum in bronchial asthma is, perhaps, more characteristic than 
that of any other pulmonary condition. During the paroxysm of asthma there 
may be no sputum, or it may be scanty, consisting of the glairy mucoid plugs 
known as the pearls of Laennec. The sputum contains many eosinophile cells 
and many alveolar epithelial cells with myelin degeneration. The mucoid 
sputum usually contains large numbers of the spirals of Curschmann along 
with the Charcot-Leyden crystals. In some cases of asthma one finds small 
cylindrical casts of bronchi. Some of these branch while the majority are 
straight and may taper at one end into the central fiber of a true spiral. 

(7). Influenza. 

The sputum of the pulmonary type of this condition shows in the early 
stages as a very scanty tenacious expectoration. Later it increases in amount, 
becomes muco-purulent and often blood-streaked, and is greenish-yellow in 
color. This sputum contains large numbers of Pfeiffer's bacilli, which have 
been previously discussed. 

(8). Gangrene of the Lung. 

The sputum in this condition is very profuse, is greenish-brown in color, 
is very offensive in odor, and is extremely fluid in character. It contains shreds 
of elastic tissue which serve to distinguish it from the sputum of putrid bronchi- 
tis or bronchiectasis. This sputum separates, as do other forms of sputum 
which have undergone stagnation and decomposition, into three distinct layers. 

Microscopic examination shows fragments of necrotic tissue varying 
from very minute particles to those several cm. in length. Very few epithelial 
cells or leucocytes are found, but red blood-cells are more or less frequent. The 
bacterial content is usually very high, but nothing characteristic is found among 
these organisms. 

(9). Abscess of the Lung. 

The most characteristic feature of true abscess of the lung or of liver 
abscess which has perforated into the lung is the sudden appearance of a large 
amount of pure pus containing fragments of lung tissue This material usually 
has the normal odor of pus, but may become offensive, although never as mark- 
edly so as in gangrene or putrid bronchitis. The sputum of the perforating 
liver abscess is usually distinguished from that of the true lung abscess by the 
so-called "anchovy-sauce" appearance. The color may vary, due to the 
presence of various types of bile pigment. Microscopically, bilirubin crystals 
may be found. 
3 



34 DIAGNOSTIC METHODS 

(10). Perforating Empyema. 

The sputum of such conditions is composed almost entirely of pus and is 
thin and liquid. It contains many hematoidin crystals, but very little elastic 
tissue fiber or other tissue fragments. The odor is usually described as that of 
old cheese in the beginning, but soon becomes offensive owing to decomposition. 

(n). Pneumonoconioses. 

The sputum in these various conditions will depend upon the pigment 
with which the lung has been infiltrated. The expectoration is usually muco- 
purulent, very profuse, and is laden with coal-dust (anthracosis), iron-dust 
(siderosis), with stone-dust, chalk-dust or plaster of Paris (chalicosis), and with 
starch granules (amylosis). 

BIBLIOGRAPHY. 

i. Andre. La grippe ou influenza. Paris, 1908. 

2. Aufrecht. Die Lungenentziindungen. Wien, 1901. 

3. Cornet. Die Tuberkulose. Wien, 1901. 

4. De Jong. Etude histo-chimique et cytologique des crachats. Paris,^i90 7. 

5. Hoffmann. Die Krankheiten des Bronchien. Wien, 1900. 

6. Sticker. Lungenblutungen. Wien, 1901. 



PLATE III. 




Leptothrix and Spirocheta Buccalis. (Unstained Specimen.) 



CHAPTER II. 
ORAL, NASAL, AURAL, AND CONJUNCTIVAL SECRETIONS. 

I. Oral Secretions. 

(i). General Considerations. 

The oral secretion is a mixture derived from the various buccal glands, 
the submaxillary, sublingual, parotid, and mucous glands. To this secretion 
has been given the name saliva. It is a colorless, odorless, and tasteless fluid, 
which appears somewhat stringy and frothy, separating on standing into two 
layers, the upper one of which is clear and the lower one cloudy. The function 
of this secretion is to moisten the mouth and throat and, also, to aid in swallow- 
ing the food as well as partially to digest the starchy food through the action 
of a specific ferment (ptyalin) which it contains. The normal daily amount 
of saliva secreted is usually about 1,500 c.c, this quantity varying under the 
influence of many factors, both physiologic and pathologic. The specific 
gravity ranges between 1,002 and 1,009 gi ym g a tota l s °lid content of 3 to 12 
grams. Its reaction is alkaline, corresponding to 0.006 to 0.048 per cent. 
of sodium hydrate. While the reaction of the saliva is normally always alkaline, 
we occasionally find an acid reaction, especially in children and in the early 
morning hours, due to the production of lactic acid by the bacteria which are 
always present in the mouth. Likewise we find an acid reaction especially in 
conditions associated with acidosis, such states very frequently leading to dental 
caries and to many other irritative conditions of the mouth. The recent work 
of Talbot along this line is especially interesting. 

The chemical composition of the saliva does not have any great clinical 
significance 1 with the exception of the presence of the sulphocyanates, the nitrites, 
and the characteristic ferment ptyalin. These substances seem to have some 
importance both from a diagnostic and symptomatic standpoint, so that a few 
remarks may be timely. The presence of potassium sulphocyanate (KCNS) 
is more or less characteristic of normal saliva and may be detected as follows: 
Collect a few c.c. of saliva before meals and allow this to filter. Add a few 
drops of hydrochloric acid and then a drop or two of ferric chlorid solution, 
when a distinct red color will be observed, whose depth will depend upon the 
amount of sulphocyanate present. It has been stated that heat should be ap- 
plied in this test, but the writer has never found it necessary as the characteristic 
reaction almost invariably appears in the cold. This color disappears on the 
addition of mercuric chlorid solution, which fact may serve to differentiate 
it from the similar one given by the saliva of opium habitues and due to meconic 
acid. Very little pathologic significance has been attached to variations in 

1 See Herzfeld and Stocker (Zentralbl. f. inn. Med., 1913, XXXIV, 753) for a discussion of 
uric acid in saliva. 

35 



36 DIAGNOSTIC METHODS. 

the amount of the sulphocyanate in the saliva, but it is interesting to note 
that in many cases of diabetes as well as in cases of severe stomatitis this sub- 
stance is frequently absent. 

The nitrites may be detected by the more delicate tests used in water analy- 
sis, as their amount is usually not sufficient for the ordinary qualitative tests. 
A very good test is the use of the Griess-Ilosvay reagent (1/2 gram of sulphan- 
ilic acid is dissolved in 150 c.c. of dilute acetic acid and treated with 1/10 gram 
of naphthylamin dissolved in 20 c.c. of boiling water. On standing, a blue 
sediment forms which is separated and dissolved in 150 c.c. of dilute acetic 
acid). On treating 10 c.c. of saliva with a few drops of this reagent and heating, 
a red color will develop in the presence of nitrites. 

The most important constituent of saliva is the ptyalin which has a defi- 
nite hydrolytic action upon starch, converting this polysaccharid into maltose 
through the intermediate stages of erythrodextrin and achroodextrin. This 
action may be readily seen by treating a little starch paste with a few c.c. of 
filtered saliva and placing the vessel in the incubator for 10 to 15 minutes. 
At the end of this time iodin solution is added when a distinct red color or an 
entire absence of color will be noticed. It is to be remembered here that starch, 
treated with iodin, is colored blue, so that a change of the starch is evident by 
the color reaction. Careful work by Litmanowicz l has shown that the dias- 
tatic power of saliva is unaffected by physiologic or pathologic variations in 
general body functions. 2 

(2). Microscopic Examination. 

On allowing saliva to stand it separates into two distinct layers, the upper 
one clear and containing the liquid portion, while the lower is cloudy and con- 
tains the morphological elements. In the microscopic examination of this lower 
layer we observe many epithelial cells in the form of large, irregular, squamous 
cells which are derived from the mucous membrane of the mouth and tongue. 
The number of these cells present depends, of course, upon the erosion to 
which the mouth has been subjected by various irritants either of the food or of 
disease. The characteristic cells of the saliva are the salivary corpuscles, 
which resemble the leucocytes, but are larger and more granular. Occasion- 
ally red blood-cells are seen, but these have no direct significance other than to 
denote ulcerative or markedly irritative conditions somewhere in the naso- 
pharynx. Beside these constituents of the saliva, we find, microscopically, 
many micro-organisms of the mold, yeast, and bacterial types. The bacteria 
are always present in the mouth as they are taken in with the air, food, and drink. 
Few of these have any direct significance, although the spirochaeta buccalis 
and microdentium should be borne in mind, especially when an examination 
is being made for the spirochaeta pallida. 3 The former is differentiated from 
the latter by the fact that its ends lie upon a line drawn longitudinally through 
the center of its spirals, while such a line drawn through the pallida lies above 

1 Zentralbl. f. d. ges. Physiol, u. Path, des Stoffw., 1909, IV, 81. 

2 See Hirata, Biochem. Ztschr., 1912, XLVII, 167; van Haeff, Nederl. Tydschr. v. Geneesk., 
1915, LIX, 307; Lubimoff, Russk. Vrach., 1915, XIV, 269. 

3 See Thibaudeau, Jour. Am. Med. Assn., 1912, LIX, 446. 



ORAL, NASAL, AURAL, AND CONJUNCTIVAL SECRETIONS. 37 

and below its ends. Moreover, it should be remembered that the smegma 
bacillus is an occasional habitant of the mouth and throat and may occur in 
specimens of sputum, giving rise to the assumption of the presence of tubercle 
bacilli unless proper means of identification are used. Simon has pointed out 
an interesting fact that the majority of the micro-organisms which are constantly 
present in the mouth cannot be cultivated on artificial media, while the temporary 
invaders easily develop. Many pathogenic bacteria have been found in the 
mouth of the healthy subject. 1 This is interesting clinically as showing the 
constant danger to which we are all subject, in case our resistance becomes low- 
ered. The writer recalls that the most virulent culture of pneumococci obtained 
from 200 throats, both diseased and normal, was from his own at the time when 
he was in perfect condition and showed no symptoms thereafter. Beside the 
pneumonia organism, streptococci and diphtheria bacilli are frequently found 
in the mouths of perfectly healthy individuals. Molds and yeast fungi are rarely 
found in the saliva during health, but they are frequently present in pathological 
conditions. 

(3). Pathologic Changes. 

The normal daily secretion of the saliva is, as stated above, about 1,500 c.c. 
The composition of the secretions of the various glands, which contribute to 
the mixed secretion, differs rather widely, the one from the other. We may, 
therefore, have changes, not only in amount of saliva, but, also, in the quality, 
depending on the diseased condition of one or more of these glands. The 
quantity of saliva is diminished in inflammation of the salivary glands, such as 
in parotitis, in all febrile diseases, in diabetes, and in nephritis. The secretion 
is also diminished by the therapeutic use of preparations of belladonna and of 
opium. It is increased by certain poisons, such as pilocarpin and mercury, 
by excessive irritation with acids and alkalies, and, also, by irritations arising 
from carious teeth. Occasional cases have been reported of a greatly increased 
amount of saliva through some obscure nervous reflex, while such a condition is 
not unusual in pregnancy. An increased flow of saliva is known as salivation 
or ptyalism. In determining whether or not salivation really exists, obser- 
vation will frequently show increased amounts of saliva at all times. In some 
cases, however, it is necessary to measure the amount and, also, to make later 
chemical examinations of the saliva. The best way of obtaining saliva free 
from contamination is to wash the mouth thoroughly with a solution of sodium 
bicarbonate, brush the teeth thoroughly with the same solution, and then rinse 
out the mouth with cold water. On now touching the inner surface of the 
teeth or the edge of the tongue with a glass rod that has been dipped into dilute 
acid, saliva will be seen pouring into the mouth from many points. This saliva 
is then collected in clean receptacles and the quantity measured. 

Variations in the reaction of the saliva are not uncommon in pathologic 
conditions. In various intestinal diseases with which we may have an associ- 
ated stomatitis, an acid reaction is frequently noted. Also in fevers, diabetes, 
starvation, and other conditions giving rise to acidosis (overloading of the system 

1 See Kiister, Kolle and Wassermann's Handbuch, 19 13, VI, 435. 



38 DIAGNOSTIC METHODS. 

with acid products) the reaction of the saliva is always acid. Strauss and 
Cohn believe that the saliva is practically always alkaline, even under pathologi- 
cal conditions. 
Coating of the Tongue. 

A coating of the tongue is practically always abnormal, as the normal 
appearance is a bright reddish color with no visible deposits. 1 A change in the 
normal appearance of the tongue has so long been indicative, in the minds 
of the profession, of disturbed conditions not only in the mouth, but in the 
stomach and the bowels, that one should always take into consideration any 
such change. In severe infectious fevers a brownish coating with a furred 
appearance is practically always seen. This consists of remnants of food and 
of incrusted blood, along with large numbers of micro-organisms and dark 
desquamated epithelial cells. The white coating contains no blood and is 
more indicative of simple gastro-intestinal disturbance than is the brown 
coating. The so-called " tartar" which forms upon the teeth seems to consist 
of deposited calcium carbonate and contains many actively motile spirochete 
as well as large segmented leptothrices, along with leucocytes and epithelial cells. 
Pharyngomycosis Leptothrica. 

In many pathological conditions of the throat, such as tonsillitis, diphtheria, 
and thrush, we frequently find the tonsillar and other buccal structures covered 
with a coating which is, in many cases, a distinct membrane containing the 
pathogenic organisms in large numbers. Many perfectly normal subjects 
complain of the formation, in the tonsillar crypts, of plugs of material which 
are easily removed by pressure. These are frequently found in patients subject 
to tonsillitis, but, also, in those showing no pathological conditions of the 
tonsils and are closely related to Dittrich's plugs, which have been discussed. 

In the pyoid masses of pharyngomycosis leptothrica, one finds large 
numbers of lymphocytes, epithelial cells and long segmented fungi, the lepto- 
thrices buccalis, which are colored bluish-red by a solution of iodopotassic 
iodid. In such conditions the polynuclear neutrophiles are present in only 
small numbers. In some cases patches of these fungi extend over quite an area 
of the tonsils so that the appearance may be one of the formation of a diph- 
theritic membrane, although microscopic examination will at once clear up the 
diagnosis. 2 
Diphtheria. 

One of the most important examinations of the oral cavities consists in 
the detection of the diphtheria bacillus (Klebs-Loffler bacillus), as an early 
diagnosis of this disease frequently enables the physician to institute antitoxin 
treatment. Such an examination should never be omitted in any case of 
suspected sore throat, especially where any membranous patches are present. 3 
By means of a stout platinum loop or a swab of cotton a piece of membrane or a 
portion of the exudate is scraped from the throat. This material is then spread 

1 See Schilling, Deutsch. Arch. f. klin. Med., 1914, CXIII, 622. 

2 Conlon (Jour. Am. Med. Assn., 1913, LX, 900) has reported the aspergillus niger as a 
causative factor in infection of the pharynx. 

3 See Neisser and Gins, Kolle and Wassermann's Handb. d. path. Mikroorg., 1912, V, 931. 



PLATE IV. 



,V«. 















Vd+hdrine-Xill. 



Diphtheria Bacilli Showing Polar Staining. (Neisser Method, Counter Stained 

with Safranin.) 



ORAL, NASAL, AURAL, AND CONJUNCTIVAL SECRETIONS. 39 

over the surface of Loffler's blood serum and is allowed to incubate at 37 C. 
for six to eight hours. This period of incubation is of some importance as it 
has been definitely shown that at the end of six to eight hours the diphtheria 
organism is the only one which will attract much attention, while if left for a 
longer time, other organisms, especially the streptococcus and staphylococcus, 
will so far outgrow the diphtheria bacillus that this latter may be unrecognizable 
unless the incubation be carried 36 hours, when the diphtheria bacillus then 
assumes the ascendency. From this culture, cover-glass preparations are then 
made and stained for one to five minutes in Loffier's alkaline methylene blue 
solution. They are then rinsed in water, dried, and examined with the im- 
mersion lens. 

Neisser's Stain. 

This stain is supposed to differentiate the diphtheria bacillus from all 
others. The smear is stained for five minutes with a methylene blue solution 
(methylene blue, Grubler, 1 gram, 20 c.c. of 96 per cent, alcohol, glacial acetic 
acid 50 c.c, and water 950 c.c). The stain should be filtered before use. The 
specimen is heated gently during the staining process and the dye renewed as 
the stain evaporates. Wash in water 1 and stain for two minutes with an aqueous 
solution of Bismarck brown or, better, a dilute solution of safranin. The 
polar bodies will be stained a deep blue, while the body of the bacillus will 
take a light brown or red color. 2 

Microscopically, the stained organism appears as a slightly curved rod, 
but especially characteristic are the bizarre forms, such as rods with alternate 
staining and nonstaining portions, rods with distinct deeply staining polar 
bodies, club-shaped or " narrow-waisted" rods, many of which lie together in 
distinctly parallel lines. 

Diphtheria bacilli may be found in the throat for weeks after all symptoms 
have disappeared 3 so that it is wise to enforce isolation of the patient until a 
negative examination for these organisms is obtained. In causing the dis- 
appearance of these bacilli from the throats of "carriers" the method of spray- 
ing the nose and throat every three hours with a 24-hour bouillon culture of 
staphylococcus aureus, as advanced by Schiotz (Ugesk. f. Laeger, 1909, LXXVI, 
1373), has proven very satisfactory. However, Bissell (Jour. Am. Med. Assn., 
1913, LXI, 1393) believes the method is questionable as it leads to false security 
owing to the fact that the few diphtheria bacilli actually left in the deeper struc- 
tures are masked by the numerous organisms of the sprayed culture. 4 

Occasionally in examination of smears from the throat, true diphtheria 

1 Gins (Deutsch. med. Wchnschr., 1913, XXXIX, 502) advises a short (2 to 3 seconds) 
treatment at this point with Lugol's solution containing 1 per cent, of lactic acid. See, also, 
Tamura, Ztschr. f. physiol. Chem., 1914, LXXIX, 289; Nielsen, Hospitalstid., 1915, LVIII, 
225. 

2 See Raskin, Deutsch. med. Wchnschr., 191 1, XXXVII, 2384; also, Ponder, Lancet, 
1912, CLXXXIII, 22; Kinyoun, Am. Jour. Pub. Health, 1915, V, 246; Mood, Southern Med. 
Jour., 1 91 5, VIII, 482. 

3 See Markl and Pollak, Wien. klin. Wchnschr., 1913, XXVI, 1617. 

4 See Womer, Jour. Am. Med. Assn., 1913, LXI, 2293. Hektoen and Rappaport (Jour. 
Am. Med. Assn., 1915, LXIV, 1985) advise the use of kaolin, either swallowed or given as an 
insufflation, for this purpose. 



4Q 



DIAGNOSTIC METHODS. 



bacilli may be confounded with pseudo-diphtheria bacilli, and in examination 
of other specimens, such as those taken from the eye, the bacillus xerosis may 
be confusing. These different organisms are best differentiated by the study 
of their action in fermenting or not fermenting certain sugars. According to 
Knapp, the pseudo-bacilli will ferment none of the sugars, the diphtheria bacilli 
will ferment dextrose, mannite, maltose, and dextrin, but not saccharose, while 
the xerosis bacillus ferments dextrose, mannite, maltose, and saccharose (cane 
sugar), while it does not ferment dextrin. The details of the methods for these 
tests must be found in bacteriological works. (See Chapter XI.) 

Vincent's Angina (Ulceromembranous Angina and Stomatitis). 

In this condition, smears taken from the throat, as well as the free saliva 
will be found to contain many organisms of two especial types, the first, spirilla, 
and the second, long fusiform bacilli. Usually both of these types are found 
together, but occasionally the spirilla are absent. The spirilla usually measure 
from 36 to 40 microns in length and 1/2 micron in breadth, while the bacilli 



^M 




'•■ A 






&»' 




Fig. 12. — Vincent's spirillum and bacillus. (Coplin.) 

are 6 to 12 microns in length and are somewhat thicker in the center than at 
the end. These organisms may be readily stained with Loffler's methylene 
blue, gentian violet, or dilute carbol-fuchsin, but they decolorize with Gram's 
method. 1 They are regarded by some as representing definite stages in the 
development of a trypanosome. 

Streptococcic Sore Throat. 

During recent years several milk-borne epidemics of sore throat with severe 
constitutional symptoms have occurred, especially in Boston, Chicago and Balti- 
more, of which the causative organism is a peculiar streptococcus (the strep- 
tococcus epidemicus). In smears from the throat and tonsillar exudates this 
highly virulent organism occurs in short chains, the spherical cocci appearing 
in twos in the chain. They are strongly Gram-positive and are surrounded by 

1 The organism has been recently cultivated, but inoculation experiments have been un- 
successful. See Gins, Kolle and Wassermann's Handb. d. path. Mikroorg., 1912, V, 1003. 



ORAL, NASAL, AURAL, AND CONJUNCTIVAL SECRETIONS. 4 1 

a definite capsule. They produce a relatively narrow zone of hemolysis on 
blood agar but little or no greenish color. 1 

Gonorrheal Stomatitis. 

In this condition the usual changes of infection are observed along with 
the appearance of the gonococci in the smears. Boston reports several cases 
of supposed gonorrheal stomatitis in which cultural methods showed the 
absence of this organism, although the smears showed the presence of intra- 
cellular Gram-negative diplococci. Such reports are not surprising in view 
of the fact that so many saprophytic diplococci (such as the diplococcus flavus 
and crassus) are found which may or may not stain by Gram's method. The 
almost constant presence (in the throat) of the micrococcus catarrhalis must be 
borne in mind. 
Thrush. 

This is a condition most commonly seen in children, but may occur in 
adults, especially in those with tubercular tendencies. The saliva in this con- 
dition is usually acid and somewhat increased in amount. Microscopic ex- 
amination of the membrane shows many epithelial cells, leucocytes, and much 




Fig. 13. — Oidium albicans. {Kolle and Wassermann.) 

granular detritus with a network of branching band-like formations, showing 
distinct segments. 2 The contents of the segments are clear and usually contain 
two highly refractive granules, one at each pole. This organism is known as 
the Oidium albicans. It stains well with the ordinary aqueous methylene 
blue solution. 

1 See Davis and Rosenow, Jour. Am. Med. Assn., 1912, LVIII, 773; Hamburger, Ibid., p. 
1109; Davis, Ibid., pp. 1283 and 1852; Capps and Miller, Ibid., p. 1848; Heinemann, Ibid., 
1912, LLX, 716; Leutscher, Ibid., p. 869; Frost, Pub. Health Reports, 1912, XXVII, 1889; 
Stokes and Hachtel, Ibid., p. 1923; Winslow, Jour. Infect. Dis., 1912, X, 73; Hamburger, Bull. 
Johns Hopkins Hosp., 1913, XXIV, 1; Capps, Jour. Am. Med. Assn., 1913, LXI, 723; Mann, 
Jour. Infect. Dis., 1913, XII, 481; North, White and Avery, Ibid., 1914, XIV, 124; Capps 
and Davis, Jour. Infect. Dis., 1914, XV, 130; Arch. Int. Med., 1914, XIV, 650; Rosenow and 
Moon, Jour. Infect. Dis., 1915, XVII, 69; Kirkland (Brit. Med. Jour., 1914, I, 419) reports 
a case from Cheltenham which belongs in this group; Bray, Jour. Am. Med. Assn., 1915, 
LXIV, 1 127. 

2 Ashford (Jour. Am. Med. Assn., 1915, LXTV, 810 and 1893) reports the finding, in most 
cases of this type, of a monilia (yeast) not hitherto described. 



42 DIAGNOSTIC METHODS. 

Oral Endamebiasis. 

The condition, known as pyorrhea alveolaris or Riggs' disease, has for years 
been regarded as a form of bacterial infection in which local irritative processes 
and systemic disturbances are looked upon as predisposing factors. Many 
types of bacteria, such as streptococci, pneumococci and staphylococci, have 
been found and isolated from the pus pockets about the roots of the teeth, but 
these cannot be regarded as the only etiologic factors in this condition, at least 
in the majority of cases. 1 

While it has been known for years that parasites, especially those of the 
ameba group, were very frequently found in the mouth and in the soft tartar 
about the teeth, these were regarded as innocuous invaders, except in a few 
isolated cases. During the last year a great amount of work, following the first 
communication of Barrett, has centered about these amebae and their associa- 
tion with the lesions of pyorrhea, until at present their pathogenic importance is 
being freely discussed. There is little question, according to Smith and Barrett, 
that we must now "regard these parasites as either directly causative of a large 
class of gingival and alveolar pyorrheas or as important members of a symbiotic 
chain with one or other of the numerous associated vegetable micro-organisms, 
in the production of these lesions." It is to be said that the proof of the 
pathogenic importance of the endamebae in Riggs' disease rests upon their al- 
most constant presence in the suppurating pockets of pyorrhea (while they are 
rarely found in healthy mouths), and the prompt removal of both suppuration 
and of the endamebae when emetin (first advocated by Roger) is administered 
locally or generally. The organisms have not yet been cultivated, so that the 
question of conformity with the classic postulates of Koch has not been in- 
vestigated. Further, many of the cases supposedly cured by emetin show re- 
currences after a few weeks' freedom from treatment. While there is no 
question of the presence of these amebae in cases of pyorrhea, it has not been 
firmly established, as yet, that they are truly pathogenic rather than merely 
" members of the symbiotic chain." It is evident, therefore, that more detailed 
study is necessary before the question of pathogenicity is settled. 

Several forms of endamebae have been described as habitants of the buccal 
and dental tissues, some of which were supposed to be pathogenic. These 
various amebae are more or less similar in their general characteristics, so that 
it is more than probable that the one, now first recognized by Barrett and a little 
later by Bass and Johns as almost constantly associated with pyorrhea, is the 
same as was long ago described by Gros, Steinberg, Grassi, Flexner and Pro- 
wazek. It is no more than just, therefore, that this ameba, whose pathogenicity 
is being investigated, should be styled Endameba 2 gingivalis (Gros) and not Enta- 
moeba buccalis (Prowazek), as some call it. 

These endamebae, as studied by Smith and Barrett, show the following 
characteristics: "Naked parasitic amebae of usual diameter in resting examples 

1 See Nodine, Dental Cosmos, 1914, LVT, 969; Gilmer and Moody, Jour. Am. Med. 
Assn., 1914, LXIII, 2023; Billings, Ibid., 2024; Mayo, Ibid., 2025; Rosenow, Ibid., 2026; 
Craig, Ibid., 2027; Hartzell and Henrici, Ibid., 1915, LXTV, 1055. 

2 The name endameba was first suggested by Leidy in 1879 and should take precedence of 
the term entameba advocated by Casigrandi and Barbagallo in 1897. 



PLATE V. 




Camera Lucid a Drawings of Endameba Gingivalis (Gros), Stained with Iron Hematoxylin. 

(A. J. Smith and M. T. Barrett, Jul. of Parasitology.) 



ORAL, NASAL, AURAL, AND CONJUNCTIVAL SECRETIONS. 43 

of 0.030 to 0.035 mm * (with exceptional instances reaching 0.040 or slightly 
above) ; with refractile and faintly greenish- tinted hyaline ectosarc well defined 
from the granular endosarc, but sometimes so thin as to be easily overlooked; 
endosarc granular, colorless and in all but the more minute examples containing 
few to many digestion vacuoles in which globular detritus of leukocytic nuclei 
and red blood cells are commonly found along with bacteria; with a small 
(0.002 to 0.005 nim.) rounded nucleus, invisible or at best uncertainly distinguish- 
able in the unstained specimen, usually central or subcentral in position, but at 
times eccentric (when the ingesta push it to one side). The nucleus is very 
poor in chromatic substance, vesicular, with a small ' binnenkorper,' some- 
times showing a minute centriole, a clear space between it and the nuclear border 
containing no chromatin or at most a very few incomplete threads; and the 
border is represented by a thin but somewhat irregular line of chromatin, about 
which the writers are unable to recognize a further membrane. The degree of 
motility manifested by the parasite is fairly comparable to that of the dysenteric 
endameba. The pseudopods as a rule are few (one, two or three), usually 
broadly lobose and commonly attaining a maximum length of the diameter of 
the endosarc. The pseudopods are composed practically entirely of the ecto- 
sarc, the granular endosarc terminating at the base or extending but a little 
into the larger ones." 

The material for examination is best obtained by scraping the bottom 
of the lesion in the peridental membrane between the tooth and alveolar process, 
as the endamebae do not live on the normal gum margin but at the edge of the 
tissue which they are invading. As the secondary infection with pyogenic 
organisms causes a profuse flow of pus which distends the pocket, very few end- 
amebae can reproduce and live in this necrotic material. For this reason it is 
absolutely essential for success in this examination that most of this purulent 
material be removed from the old lesions and the specimen for examination be 
obtained from the bottom of the lesions. By means of a toothpick or, preferably, 
a Younger scaler No. 8 or 9, remove some of the material from the bottom of the 
lesions. If the specimen is to be examined in the fresh condition, mix the 
material with a drop or two of slightly warm normal saline solution on a warm 
slide and place over this a cover-glass. Examine at once with the high-power 
dry lens. Careful study will reveal the motile organisms showing the charac- 
teristics mentioned above. If the organisms are not actively motile, it is not the 
simplest matter to recognize them in the fresh state unless one is fairly familiar 
with them. 

For general work the stained specimen is, probably, more satisfactory for 
examination, as differentiation is more certain. Obtain the material as above 
mentioned and scrape it onto a clean slide with a toothpick, spreading it out 
into a thin layer with the flat side of the toothpick. Care should be taken not 
to rub the material back and forth as this may break up the amebae. Allow the 
slide to dry in the air and fix by passing once or twice through the flame. Cover 
the smear with carbol-f uchsin for 1 o to 1 5 seconds, wash off the excess of stain 
with water and stain with Loffler's methylene blue for 30 seconds. Wash in 
water, dry and examine with the immersion lens. Properly prepared specimens 



44 DIAGNOSTIC METHODS. 

should have a deep purple color. The red blood cells stain a deep red. Pus 
cells show a bright purple nucleus with a light pink protoplasm. Tissue cells 
are larger than the pus cells and appear reddish with a small purple nucleus. 
Bacteria may be stained either red or blue, depending on their type. The 
endamebae vary in size from cells somewhat smaller than a pus cell to those several 
times as large. The endoplasm stains a deep blue, while the irregular border of 
ectoplasm stains purple. A small round or oval centric nucleus is observed 
staining a deep port- wine color, while many inclusion bodies (contained in 
vacuoles) may be seen staining a deep purple. Usually the entire endameba 
appears to be surrounded by a clear zone, due to retraction during drying. 1 

II. Nasal Secretion. 

This secretion does not present many points for study and seems to be of 
pathologic significance only in infectious conditions. Normally, the nasal secre- 
tion is comparatively scanty, clear, tenacious, odorless, salty in taste, and alka- 
line in reaction. It is largely composed of mucus, showing squamous and cili- 
ated epithelium in abundance, 2 occasionally leucocytes, large numbers of 
bacteria and Charcot-Leyden and triple phosphate crystals. The bacterial 
content of the nasal secretion is made up of both pathogenic and non-pathogenic 
organisms, the tubercle bacilli having been frequently obtained from a nor- 
mal mucous membrane. 3 
Pathologic Changes. 

In most acute infections, as well as in the so-called acute colds, the nasal 
secretion is at first diminished in amount, but soon becomes very profuse. 
This secretion shows the same appearance as does the normal fluid, but as ulcera- 
tion ensues may be heavily loaded with pus-cells and bacteria. The chronic 
suppurative process in the nose may affect any or all of the accessory sinuses, 
so that we may have very severe conditions arising from simple ulceration. 
Frequently the ulcerative and membranous conditions spoken of above may ex- 
tend from the mouth to the nose, so that distinct diphtheritic membranes 

1 Gros, Bull. Soc.imp.nat.de Moscow, 1849, XXII, 549; Steinberg, Souremenaya meditsina, 
Dissert. Kiev, 1862; Grassi, Gazz. med. Ital.-Lomb., 1879, XXXIX, 446; Flexner, Bull. Johns 
Hopkins Hosp., 1892, III, 104; Kartulis, Ztschr. f. Hyg., 1893, XIII, 9; Prowazek, Arbeiten 
a. d. Kais. Gesundheitsamte, 1904, XXI, 42; Leyden and Lowenthal, Charite Ann., 1905, 
XXIX, 3; Verdun and Bruyant, L'Echo med., du Nord, 1907, XI, 375; Bruyant and Pelissier, 
Ibid., 1909, XIII, 301; Brumpt, Precis de Parasitol., Paris, 1910; Barrett, Dental Cosmos, 
1914, LVI, 948 and 1345; Chiavaro, Editorial in Dental Cosmos, 1914, LVI, 1089; Austral. 
Jour. Dent., 1914, XVIII, 343; Dental Review, 1914, XXVIII, 1122; Bass and Johns, New 
Orleans Med. and Surg. Jour., 1914, LXVII, 456 and 671; Smith, Middleton and Barrett, 
Jour. Am. Med. Assn., 1914, LXIII, 1746; Evans and Middleton, Ibid., 1915, LXTV, 422; 
Bass and Johns, Ibid., 553; Howe, Dental Cosmos, 1915, LVII, 307; Brit. Dental Jour., 1915, 
XXXVI, 464; Talbot, Dental Cosmos, 1915, LVII, 485; Jour. Am. Med. Assn., 1915, LXTV, 
928; Rogers, Indian med. Gaz., 1915, I, 121; Stewart, Med. Record, 1915, LXXXVII, 798; 
Dental Cosmos, 1915, LVII, 605; Sturridge, Ibid., 780; Johns, Interstate Med. Jour., 191 5, 
XXII, 529; Smith, Jour. Am. Med. Assn., 1915, LXIV, 1567; Smith and Barrett, Jour. Para- 
sitol., 1915, I, 159; Brunelles and Ginsberg, New York Med. Jour., 1915, CII, 554; Williams, 
von Shelly and Rosenberg, Proc. New York Path. Soc, 1915, XV, 334; Bass and Johns, 
Alveodental Pyorrhea, Saunders, Phila., 1915; Hecker, Am. Jour. Med. Sc, 1915, CXLLX, 889. 

2 Proskauer (Berl. klin. Wchnschr., 1914, LI, 172 and 255) has shown that some of the 
cells may be melanotic. 

3 See Kiister, Kolle and Wassermann's Handb. d. Path. Mikroorg., 1913, VI, 450. 



ORAL, NASAL, AURAL, AND CONJUNCTIVAL SECRETIONS. 45 

are frequently found in the nasal cavities. Molds may develop to such an 

extent that true pathological lesions obtain. 1 

Rhinitis. 

At present the infectious nature of the catarrhal conditions known as " colds" 
is generally accepted, many organisms, such as micrococcus catarrhalis, 
pneumococcus, streptococcus, influenza bacillus and others, having been as- 
sociated in different cases with the etiology. It would seem that these organisms 
are, however, to be regarded as simply secondary invaders, in many cases at 
least, and become of importance only when the discharge is purulent. 

Tunnicliff 2 has found, in the mucoid stages of acute and chronic rhinitis, a 
delicate, curved, Gram-negative bacillus, which appears to have a direct patho- 
genic association with these conditions. While this Bacillus rhinitis of Tunnicliff 
is found in only 6 per cent, of normal noses, it was present in 98 per cent, of 
cases of acute rhinitis and in 90 per cent, of cases of chronic non-purulent 
rhinitis. It is, apparently, not found at all in purulent rhinitis, the other 
organisms mentioned above being the causative factors in this latter condition. 
There is little doubt that this organism has an etiologic relationship to acute 
and chronic rhinitis on account of its almost constant presence in the nose in 
such cases, its general absence from the normal nose, its ability to produce 
rhinitis experimentally with recovery in pure culture, and on account of the 
production, in cases of acute and chronic rhinitis and in persons injected with the 
bacillus, of specific opsonins and complement-binding bodies. 

This organism may be found by spreading a fairly large amount of the 
nasal discharge (mucus) on a glass slide, drying and staining with carbol-fuchsin. 
Often a large number of microscopic fields may have to be examined before the 
organisms are found. They vary in length from 5 to 8 microns and from 1/3 
to 1/2 micron in width. The ends are pointed or slightly rounded; generally 
the bodies are slightly curved, but may be straight, wavy or bent over at one end. 
A ring and an enlargement in the form of a ball are occasionally seen at one ex- 
tremity. The organism is a strict anaerobe growing at 37 C. slowly, the 
colonies on slightly alkaline goat-blood agar appearing usually in about seven 
days as small, dull, round growths. The organism does not grow on plain 
agar. Specimens from cultures stain a little more deeply than those taken 
directly from the mucus, but they retain their Gram-negative characteristics. 
Hay Fever. 

In this condition the nasal secretion is found to be increased to a large extent 
at certain times of the day and much diminished at others, depending upon the 
paroxysms of the disease. Nothing of pathological importance has been found, 
however, in the examination of the nasal secretion in this condition, no specific 
organism having been identified. 
Meningitis. 

In some cases of meningitis the cerebrospinal fluid passes into the nasal 
cavity as a result of caries of the bones of the skull. This fluid may be dis- 

^eeTilley, Jour. Laryngol., Rhinol. and Otol., 1915, XXX, 145. 

2 Jour. Infect. Dis., 1913, XIII, 283; Ibid., 1915, XVI, 493. See Howell, Ibid., 456; 
also, Kruse, Munch, med. Wchnschr., 1914, LXI, 1547. 



4-6 DIAGNOSTIC METHODS 

tinguished by the fact that it contains practically no albumin, but does show the 
presence of a reducing substance which may or may not be sugar. This fluid 
may also contain the diplococcus intracellularis meningitidis of Weichselbaum. 
While this organism is not found in all cases of epidemic meningitis, yet it is 
found in many, so that the nasal secretion may be of some diagnostic importance 
from this standpoint. The above diplococcus is similar, both in morphological 
and staining characteristics, to the gonococcus, and presents many difficulties 
in differentiation (see cerebrospinal fluid). 

In the course of glanders, leprosy, plague, pneumonia, typhoid fever, 
influenza, and many other infectious diseases, the characteristic organisms of 
these conditions may be found in the nasal secretion. Recently Goldberger 
and Anderson 1 have demonstrated the presence of the unknown virus of 
measles in the mixed buccal and nasal secretions. 2 

Occasionally concretions are found in the nose, but these rarely reach a 
large size and do not have very great pathologic significance. They are largely 
composed of vegetable fibers taken in by inhalation and cemented by mucus 
which is hardened by the deposition of lime salts. 

In the condition known as ozena, the nasal secretion and fetid crusts, so 
characteristic of this disease, are found to contain numerous organisms of various 
types, among which one observes a peculiar cocco-bacillus which is difficult 
of cultivation and isolation. This organism was first noted by Perez, but it was 
not until the later studies of Hofer and his associates that it was practically 
established as the etiologic factor in this disease. To this micro-organism has 
been given the name coccobacillus foetidus ozence, (Perez). 3 

III. The Aural Secretion. 

Normally no secretions appear in the external ear, with the exception of 
that of cerumen, while the secretion of the middle ear and of the internal ear 
is normally inaccessible to examination. We find, therefore, that the chief im- 
portance which is attached to the clinical examination of the aural secretions, is 
entirely a pathological one. In catarrhal and inflammatory conditions of the 
external auditory canal, one finds naturally very large numbers of organisms, 
with which the disease njay or may not be associated. In the chronic inflam- 
matory processes of the middle ear, the more important organisms found are the 
pneumococcus, streptococcus pyogenes, staphylococcus pyogenes, bacillus 
pyocyaneus, the bacillus of Friedlander, the bacillus coli communis, the diplo- 
coccus intracellularis, the typhoid bacillus, and especially the diphtheria bacil- 
lus. As disease of the middle ear is so commonly associated with disease of the 
naso-pharynx, it is possible to find in the discharge from the ear any organism 
which is causing trouble either in the nose or in the throat. Hamilton has 

1 Jour. Am. Med. Assn., ion, LVII, 476 and 971; Am. Jour. Dis. Child., 1912, IV, 20. 

2 See also Lucas and Prizer, Jour. Med. Research, 1912, XXVI, 181. 

3 See Hofer, Wien. klin. Wchnschr., 1913, XXVI, ion; also, Abel and Hallwachs, Kolle 
and Wassermann's Handbuch, 1913, VI, 534; McGowan, Jour. Laryngol., Rhinol. and 
Otol., 1915, XXIX, 57; Guggenheim, Interstate Med. Jour., 1915, XXII, 129; Horn, Jour. 
Am. Med. Assn., 191 5, LXV, 788. 



PLATE VI. 



/ 



/ 



Katharine Hill 



MORAX-AxEXFELD DlPLOBACILLES. (GRAM'S STAIN.) 

Courtesv of Dr. Brown Pusev. 



M 



wm 



Katnarm^ H il\ 



Koch- Weeks Bacillus. (Gram's Stain.) 
Courtesv of Dr. Brown Pusev. 



ORAL, NASAL, AURAL, AND CONJUNCTIVAL SECRETIONS. 47 

shown the almost constant presence of the pseudo-diphtheria bacillus in the 
discharge of the running ears following scarlet fever. 

It is not an uncommon thing to find certain inflammatory processes of para- 
sitic origin in the external auditory canal. This condition is known as otomyco- 
sis and is frequently caused by the aspergillus niger. Besides this organism, 
many other fungi belonging to this group have been found in the external ear. 
Among these we find the aspergillus flavus and fumigatus, the aspergillus nidu- 
mus, the mucor septatus, the eurotium malignum, and the penicillium minimum. 
These parasites are very readily detected by removing a small portion of the 
mycotic mass and spreading it thinly on a slide. Add a small drop of water, 
apply a cover-glass, and examine under a high-power dry lens. For the details 
of structure of such organisms, bacteriological works should be consulted. 

Besides these fungi we occasionally find in the external auditory canal larvae 
of various insects. These larvae may develop later into the full-grown insect 
and may be removed from the ear by the movements of the animal. In other 
cases such larvae have incited inflammatory processes which are occasionally 
troublesome, as in the case reported by Richardson. 

IV. The Conjunctival Secretions. 

Under normal conditions the secretion of the conjunctiva and of the lacri- 
mal gland concerns us only very little. It is in the course of an inflammatory 
process in which these secretions may be greatly increased and greatly changed 
by the inflammation that any clinical importance attaches to them. In in- 
flammatory conditions of the conjunctiva we find certain organisms which 
require identification in order that proper treatment may be instituted and the 
proper prognosis given. 1 It is to be recalled that the pseudo-diphtheria 
bacillus is practically always found in smears made from the conjunctival 
secretion, yet it is rarely, if ever, pathogenic in this situation. 

Pathologic Changes. 
Diphtheritic Conjunctivitis. 

In cases of conjunctivitis which are traceable to infection with the diph- 
theria organism, we frequently find the formation of an extensive membrane 
which consists of epithelial cells, leucocytes, and large numbers of streptococci 
along with the diphtheria bacilli. Clinically, a membrane formation on the 
conjunctiva may arise from infection of this tissue with organisms other than 
the diphtheria bacillus; hence, it is wise in all suspicious cases to submit a portion 
of the membrane to both direct microscopic and cultural examinations. The 
appearance of the organism has been previously described. 

Infectious Conjunctivitis. 

In acute infectious conjunctivitis, various organisms have been found, 
the most common ones being the Koch-Weeks bacillus, the pneumococcus, 

1 Wilder and McCullough (Jour. Am. Med. Assn., 1914, LXII, 1156) report a case of 
sporotrichosis of the eye in which the organisms were observed, in direct smears, as Gram- 
positive oval bodies and in cultures as typical sporothrices. 



48 DIAGNOSTIC METHODS. 

and the gonococcus. Occasionally one finds the staphylococcus, streptococcus, 
colon bacillus, influenza bacillus, Morax-Axenfeld bacillus, the diphtheria 
bacillus, and other organisms. 1 

In some regions the Koch- Weeks bacillus is frequently found as the etiologic 
factor in acute infectious conjunctivitis, while in others it is rarely, if ever, 
observed. This is an organism of the influenza group, a small, thin, Gram- 
negative bacillus, which is, so far as known, pathogenic only for the human 
conjunctiva (see cut). It grows best on media containing a slight amount of 
human blood, especially in symbiosis with the xerosis bacillus. This latter 
organism is differentiated from the Klebs-Loffler bacillus only by the appli- 
cation of the fermentation tests spoken of under Examination for Diphtheria 
Bacilli in the Throat. 

The most common bacterial cause of chronic conjunctivitis is the bacillus 
of Morax-Axenfeld, usually seen as a diplobacillus, several groups of which may 
at times be arranged in chains (see Plate V). It is a Gram-negative organism 
and grows well on Loffler's blood-agar, which it digests, forming on the sur- 
face, at the beginning of its growth, very characteristic small pits. 

Gonorrheal Conjunctivitis. 

This form of conjunctivitis is much more common than is generally sup- 
posed, so that the identification of the organism is of great importance. In 
the development of this type of conjunctivitis, large or small amounts of pus 
are invariably present between the folds of the conjunctiva. A portion of this 
pus may be collected by means of a cotton swab or a platinum loop and smeared 
thinly over a slide. This smear is then fixed in the flame and is stained for the 
gonococcus by both the methylene blue and Gram stains. The technic of 
this latter staining process, as well as the characteristic appearance of the 
organism, will be discussed in a later section. 

Trachoma. 

Lately, a great deal of attention has been given to some bodies, which are 
found in trachoma and which are, possibly, the long-sought cause of this infec- 
tious disease of the conjunctiva. These organisms are known as the Prowazek- 
Greefl bodies and are shown in the accompanying cut. They are best stained 
by the Giemsa stain (see Blood), smears being made in the usual manner. 

The present status of these bodies is that they are almost always found 
in the acute stages of this disease and are, occasionally, observed in other 
conditions which do not present the clinical features of trachoma. Xoguchi 
and Cohen (Jour. Exper. Med., 1913, XVIII, 572) report the cultivation of an 
organism, resembling the trachoma body, from cases of trachoma with and 
without cell inclusions. Whether or not they are the true etiologic factor must 
still be considered an undecided question. 2 

1 See Axenfeld, Kolle and Wassermann's Handbuch, 1913, VI, 545, 572 and 5S7; also, 
Noguchi and Cohen, Jour. Exper. Med., 1915, XXII, 304. 

2 See report from the Laboratories of the New York Health Department found in Proc. 
New York Path. Soc, 1912, XII, 17; Williams and associates, Jour. Infect. Dis., 1914, XTV, 
261. 



PLATE VII. 






Xafhar<f\€ H\\\ 



Trachoma Bodies of Prowazek-Greeff. (Giemsa Stain.) 
Courtesy of Dr. Brown Pusey. 



ORAL, NASAL, AURAL, AND CONJUNCTIVAL SECRETIONS. 49 

Vernal Conjunctivitis. 

An interesting point in the diagnosis of the above condition, as well as 
of the conjunctivitis of hay fever, is the fact, first observed by Herbert 1 and 
confirmed by many, that in these two types of conjunctivitis eosinophile poly- 
morphonuclear leucocytes are found in abundance, whereas in the ordinary 
forms of conjunctivitis such cells do not obtain. Pusey 2 has, therefore, been 
led to believe that these facts point strongly to a similar etiologic factor, 
namely pollen, in the vernal conjunctivitis and in the conjunctivitis of hay 
fever. 

BIBLIOGRAPHY. 

1. Axenfeld. Die Bakteriologie in der Augenheilkunde. Jena, 1907. The 

Bacteriology of the eye. New York, 1908. 

2. Davis. Les Mikrobes de la Bouche. Paris, 1890. 

3. Kohl. Die Heferpilze. Leipzig, 1908. 

4. Litchfield. Diphtheria in Practice. London, 1908. 

5. Mallory and Wright. Pathological Technic. Philadelphia, 191 5. 

6. Miller. Die Mikro-organismen der Mundhohle. Leipzig, 1889. 

7. Nuttall and Smith. Bacteriology of Diphtheria. Cambridge, 1908. 

8. Sticker. Die Bedeutung des Mundspeichels in physiologischen und 

pathologischen Zustanden. Berlin, 1899. 

1 Brit. Med. Jour., 1903, II, 73. 

2 jour. Am. Med. Assn., 1911, LVII, 1207. 



CHAPTER III. 
GASTRIC CONTENTS. 

I. General Considerations. 

The gastric juice is the product of the secretory activity of the glands of the 
stomach. Different series of glands contribute separate elements to the 
secretion, so that we find much variation, under pathologic conditions, in the 
composition of this fluid. 

The stomach should be regarded as a dilated and specialized portion of the 
general digestive tube, its walls consisting of the following four coats: mucous, 
submucous, muscular, and fibrous. From the standpoint of secretory activity 
the internal or mucous coat is the most important. This mucous membrane 
is covered throughout its entire length by a single layer of simple columnar 
epithelium. It follows the various folds or ruga dipping down in places to line 
the orifices and ducts of the tubular glands which are of such importance in the 
digestive activity of the stomach. The gastric glands are of two kinds, the 
peptic or fundus glands, situated in the middle and cardiac thirds of the 
stomach, and the pyloric glands, found in the pyloric third of the stomach. 

Peptic Glands. 

These glands are slightly wavy simple tubular depressions, in which a duct, 
a neck, and a fundus are recognizable. In exceptional cases the fundus is 
divided, while in nearly all it is tortuous or spiral its extremity being often 
sharply bent at right angles to the general axis of the tube (Piersol) . In these 
peptic glands are found two types of cell. The first, known as the central, 
chief or adelomorphous cells, bound the lumen of the gland and form the bulk 
of the glandular epithelium. These cells are either polyhedral or columnar in 
form and each contains a spherical nucleus situated within the granular pro- 
toplasm. These cells do not stain readily with aniline dyes. The chief func- 
tion of these central cells of the peptic glands is to secrete the rennin and lipase 
which are present in the gastric juice. The second type of cell in the peptic 
gland is known as the parietal, acid or oxyntic cell and is situated in the 
periphery of the gland immediately below the basement membrane. These 
cells are more oval or angular in form, are larger than the chief cells, are more 
finely granular in structure and stain deeply with the aniline dyes. They are 
directly concerned with the secretion of hydrochloric acid. • 

The Pyloric Glands. 

These glands are characterized by their relatively long wide ducts into 
which the several divisions of the body open; the tubular compartments are 
w avy and tortuous and frequently end in slightly expanded extremities. The 
duct is lined by tall columnar epithelium, the cells becoming lower and broader 

5o 



GASTRIC CONTENTS. 5 1 

as they approach the neck and toward the fundus. The cells contain finely 
granular protoplasm and do not secrete mucus but a thin albuminous liquid. 
Parietal or acid cells do not occur in the pyloric gland, being confined to the 
true peptic gland (Piersol). 

It will thus be seen that the active portions of the gastric juice are secreted 
by the fundus glands, the pyloric glands contributing nothing except a small 
amount of the ferments and liquid portion, the mucus being largely derived 
from the goblet cells which line the entire stomach and the wider portion of 
the glandular ducts. These ferments do not exist in the cells as such, but rather 
in the form of zymogens or prozymogens which become active only in the presence 
of the free hydrochloric acid. 1 

The free hydrochloric acid of the gastric juice is formed in the parietal cells 
of the peptic gland. 2 The mechanism of this formation is not absolutely estab- 
lished, but it seems probable that this free acid arises from the chlorids taken up 
from the blood by these cells. Just what is the active agent in causing the con- 
version of the chlorids into free acid seems to be in doubt, but it may be either 
. the continuous action of carbonic acid or, as Maly assumes, the interaction of the 
sodium phosphate (Na 2 HPOJ with the chlorids of the cell. It is also probable 
that the osmotic influences may be very great in the production of this free 
hydrochloric acid as Koeppe advocates. This acid is present at all times 
in the normal stomach, being found even in cases of extreme starvation. 

The careful work of Pawlow 3 has shown that various factors influence the 
quantity and quality of the normal gastric juice. He asserts that the "appe- 
tite is the first and mightiest exciter of the secretory nerves of the stomach, 
a factor which embodies in itself a something capable of impelling the empty 
stomach of the dog in the sham feeding experiment to secrete large quantities 
of the strongest juice. A good appetite in eating is equivalent from the outset 
to a vigorous secretion of the strongest juice; where there is no appetite this 
juice is also absent." Moreover, under natural conditions, the stimulation of 
food is a very important factor. The administration of a diet causes a secre- 
tion of gastric juice which is directly proportionate, both in amount and activity, 
to the diet taken. We find, according to Chigin, that the greatest digestive 
power is shown by the juice excreted after the administration of bread, although 
the total acidity is greatest following an intake of meat. If we compare equiva- 
lent weights of food material we find that flesh requires the most gastric juice 
and milk the least; but taking equivalents of nitrogen, bread needs the most and 
flesh the least. In this connection it is well to remember that the gastric secre- 
tion varies from hour to hour. Thus the most active juice occurs with flesh 
in the first hour, with bread in the second and third hour, and with milk in the 
fifth to the sixth hour. The point of all this is that the rate and time of secretion 
of the gastric juice is always characteristic for each diet. 

1 A secretagogue, called by Edkins gastrin, is believed to be formed by contact of certain 
substances with the gastric mucosa. This gastrin apparently causes a true gastric secretion 
(see Keeton and Koch, Am. Jour. Physiol., 1915, XXXVII, 481). 

2 See Harvey and Bensley, Biol, Bull., 1912, XXEII, 225; also Lopez-Suarez, Biochem. 
Ztschr., 1912, XLVI, 490; Hammett, Anat. Rec, 1915, IX, 21. 

3 The Work of the Digestive Glands. London, 1902. See, also, Wolfsberg, Ztschr. f. 
physiol. Chem., 1914, XCI, 344. 



52 



DIAGNOSTIC METHODS. 



Moreover, it has been found that the hydrochloric acid first secreted com- 
bines at once with the proteins of the various food stuffs, so that we may find 
no free hydrochloric acid in the gastric contents, although the secretion may be 
normal and may show a very high degree of total acidity. 



II. Methods of Obtaining the Gastric Contents. 

Unless the patient is one who can easily eject the contents of the stomach 
by vomiting, it is necessary to resort to the introduction of the so-called stomach- 
tube for the removal of the contents. This 
stomach-tube consists of a long, soft rubber tube 
about 75 cm. in length, having a lumen 6 to 7 
mm. in diameter and provided with either two 
oval lateral openings or with three, one being at 
the end of the tube. 1 Before introduction of the 
stomach-tube, it should be moistened with warm 
water and should be thoroughly cleaned. It is 
frequently a wise precaution, in general work 
where all classes of patients are to be examined, 
to have separate tubes which may be used by 
patients affected with tubercular or syphilitic con- 
ditions. In those patients who require frequent 
washing-out of the stomach or frequent examina- 




Fig. 14. — Stomach tube, 
tion of the stomach contents, it is wise to order separate tubes for each. 



Introduction of the Tube. 

The patient must be in a sitting posture, a towel or a rubber sheet being 
placed about his neck to prevent soiling of the clothes with the saliva or material 
which is occasionally brought up during the passage of the tube. False teeth 
should be removed and anything interfering with the passage of the tube should 
be avoided. In patients who are hypersensitive, a 10 per cent, solution of 
cocain is applied to the pharynx. The head of the patient is now bent slightly 
forward, never backward as some advise, and the mouth slightly opened, care 
being taken never to use a depressor on the tongue. The rubber tube, held as 
one would a pen, is passed gently backward over the tongue until its tip strikes 
the posterior wall of the pharynx, when it turns downward and may be readily 
introduced into the stomach, by slight forcing. As the tube reaches the esopha- 
gus, many patients complain of a sense of suffocation, which is not real but 
apparent. The tube interferes in no way with the normal respiratory move- 
ments and hence the patient should be cautioned to breathe normally and not 
forget to breathe. If the patient will swallow normally, the passage of the tube 
is greatly facilitated. It occasionally happens that highly nervous patients 
have great difficulty in swallowing this tube, so that it may be necessary to defer 

1 Rehfuss (Am. Jour. Med. Sc, 1914, CXLVII, 848) has devised a special stomach tube 
with a special tip, slotted with large perforations. This tip, on account of its weight, passes to 
the most dependent parts of the stomach and permits of more certain withdrawal of the stom- 
ach contents. See, also, Schiitz, Arch. f. Verdauungskr.. 1914, XX, 304; Lanza, Riforma 
Med., 1915, XXXI, 561. 



GASTRIC CONTENTS. 



53 



the withdrawal of the contents to a second or even a third period. It is never 
wise to excite a patient by forcing matters at any stage of the investigation. 
If any sign of cyanosis or marked pallor is evident the tube should be immedi- 
ately withdrawn and a second attempt made at some later time. When the 
tube has reached the floor of the stomach, which is in normal cases about 40 cm. 
from the incisor teeth, a distinct resistance to further passage of the tube will be 
noticed. This point should be carefully observed as the forcing of the tube 
beyond this point may produce rupture of the stomach wall or may cause the 
tube to "buckle." 1 In this latter condition it will be impossible to withdraw 
the stomach contents. Many of the tubes used for gastric examination have a 
mark indicating the normal length of tube from the incisor teeth to the stomach 
wall, so that one has a definite idea when he has introduced the tube to the 
right point. In some cases the gastric juice will commence to flow from the 




Fig. 15. — Turck's aspiration apparatus. 

tube as soon as it is properly introduced, but in the majority of cases some help 
is necessary to start the siphonage. Frequently all that is needed is to ask the 
patient to bear down with his abdominal muscles or to cough a little. In other 
cases aspiration is necessary. One may employ an ordinary Politzer bag 
or a Boas bulb for starting the fluid in the tube. This is very readily done by 
compressing the bulb and applying it, while compressed, to the end of the tube 
in such a way that the suction will be sufficient to draw the material into the 
tube. Once started, the material flows quite readily, but it may be necessary to 
use aspiration several times as the tube may become clogged with tenacious mucus 
or particles of food stuff. 

If it is desired to wash out the stomach, either to obtain the total gastric 
contents or for the purpose of mere lavage, a funnel is attached to the external 
end of the stomach-tube and about 500 c.c. of water are allowed to flow through 
the tube into the stomach. In this operation the funnel is held either on a 
level with the patient's mouth or a very little bit above. By depressing and 
inverting the funnel over a suitable vessel, before all the water has left it, 
return flow will soon set in and the stomach will be practically emptied by 

1 In this connection see Harmer and Dodd, Arch. Int. Med., 19 13, XII, 488. 



54 DIAGNOSTIC METHODS. 

siphonage. In some cases it becomes necessary to add more water, but in no 
case should any be added after the patient complains of a feeling of distress. 

In collecting the stomach contents one should avoid as far as possible any 
admixture with the saliva which is more freely excreted at this time than 
normally. This is best done by wrapping a cloth about the tube so that the 
material may be absorbed as it runs along the side of the tube. After one has 
obtained the gastric contents, the tube is compressed with the fingers and is 
rapidly withdrawn, care being taken to keep up the compression so as to hold 
in the tube material which has not already passed into the receiving vessel. 
This residual material is added to the portion first received. 

In cases in which water has been introduced to wash out the stomach after 
the gastric contents have been obtained, one should be careful to note the 
amount of fluid poured into the stomach so that he may be able to judge of the 
amount again received. 

Not every case with which the practitioner meets is amenable to such 
manipulation. We find as especial contraindications to the use of the stomach- 
tube, uncompensated valvular lesions of the heart, arteriosclerosis, aneurysm, 
advanced pulmonary tuberculosis, marked emphysema, acute febrile diseases, 
severe hemorrhage, especially from ulcer or carcinoma, and excessively devel- 
oped nervous antipathy. 

Test Meals. 

As the secretion of the gastric juice is so dependent upon administration 
of food, it has become the custom to use certain combinations of food principles, 
which will excite gastric activity and enable us to obtain a juice which will 
give us more or less definite ideas of its composition in the condition investi- 
gated. 1 It must be remembered that marked idiosyncrasy toward certain 
foods exists, so that we may not use in all cases the same sort of a diet for ex- 
citing the gastric juice. The results obtained in pathologic conditions are 
compared with those obtained from normal individuals under the influence 
of the same diet. In this way we are able to say, with some degree of certainty, 
that a suspected case shows normal or abnormal gastric relations. These 
diets, the so-called test meals, are always given to the fasting stomach and are 
removed after a suitable time by the use of the stomach-tube. The time best 
suited for the administration of these meals is in the morning, as the stomach 
has had occasion during the night to empty itself of most of its contents. 

Ewald Test Meal. 

This meal, which is, perhaps, the most frequently employed in general 
work, consists of a roll or piece of bread or toast without butter and two cups of 
water or tea 2 without milk or sugar. In approximate figures this will represent 

1 See Smithies, Am. Jour. Med. Sc, 191 5, CXLIX, 183; also, Heaton, Brit. Med. Jour. 

2 Kober, Lyle and Marshall (Jour. Biol. Chem., 1910, VIII, 95) have shown that it is 
almost impossible to detect blood in the presence of tannic and gallic acids, as found in tea. 
It is wise, therefore, not to use tea in the test meal when blood is suspected. 



GASTRIC CONTENTS. 55 

35 grams of wheat bread and 400 c.c. of water or tea. 1 The bread should be 
well masticated so that the later withdrawal of the contents may not be inter- 
fered with by the plugging up of the openings in the tube. The contents are 
removed one hour later and consist normally of 30 to 50 c.c, depending both 
upon the skill of the operator and upon the condition of the stomach. Hyper- 
motility of the stomach will diminish the quantity of contents received, while a 
hypomotility will increase the quantity. 

Boas Test Meal. 

This meal consists of a dish of oatmeal prepared by concentrating to 
500 c.c. a liter of water to which a tablespoonful of oatmeal is added. This 
meal was advised to prevent the introduction into the stomach of lactic acid 
which is a normal constituent of bread. While this small amount of lactic 
acid introduced in the Ewald meal has little significance, yet in doubtful cases 
it is well to avoid it. The contents of the stomach are withdrawn one hour 
later when the amount may be very small. If the stomach shows normal 
digestive powers most of the material will be then passed into the intestine, 
while an appreciable amount of material would indicate either a dilatation of 
the stomach or pyloric obstruction. 

Riegel Test Meal. 

This test meal has the advantage of permitting the patient to use a diet 
which is more normal than either of the ones previously mentioned. This 
diet is more important in America, where we are not accustomed to the con- 
tinental breakfasts, than it is in Germany where the Ewald and Boas meals 
are more usual. 

The Riegel meal is given in the middle of the day at a time when the 
patient is accustomed to such a meal. It consists of about 400 c.c. of soup, 
200 grams of beef-steak, and either two slices of white bread or 150 grams of 
mashed potato along with one glass of water. This meal is withdrawn at the 
end of three to four hours. It has the advantage of allowing us to judge of the 
length of time which the food remains in the stomach under normal conditions 
and, also, to form an opinion of the rate and amount of digestion which has 
taken place. This meal incites a more nearly normal gastric juice than does 
the Ewald or Boas meal, but it is such that clogging of the stomach tube by 
particles of undigested food frequently occurs. 

Fischer Test Meal. 

This meal, introduced by an American physician, has the advantage of 
more nearly approaching an American breakfast than the others. It consists 
of the bread and tea of the Ewald meal along with a quarter of a pound of 
finely chopped lean beef broiled and seasoned. The contents are removed 
at the end of three hours. 

1 Roberts (Jour. Am. Med. Assn., 1912, LVIII, 753) adds to this breakfast 30 grams of 
lactose. He extracts the meal as usual and then washes out the stomach with 200 c.c. of water. 
The total gastric juice excreted and the acidity when excreted are calculated by the method of 
Mathieu (p. 58). 



56 DIAGNOSTIC METHODS. 

Salzer Test Meal. 

This is in reality a double meal and is given as follows: For breakfast 
the patient receives 30 grams of lean cold roast meat, finely chopped, 250 c.c. of 
milk, 60 grams of rice, and one soft-boiled egg. Four hours thereafter a second 
meal is given, consisting of 35 to 70 grams of stale wheat bread and 400 c.c. 
of water. The contents are then removed one hour after this second meal. 
Under normal conditions of digestion and motility the stomach contents should 
show no remnants of the first meal. 

Sahli Test Meal. 

This meal was introduced to enable the worker to examine quantitatively 
the material withdrawn. The inconstant composition of the ordinary test 
meals makes it rather difficult to judge of the digestive power of the stomach. 
Sahli has introduced, therefore, a soup prepared as follows: Twenty-five 
grams of flour and 15 grams of butter are placed in a pan and browned over a 
fire. Three-hundred and fifty c.c. of water are then added and the whole 
boiled for five minutes (the loss in volume being replaced by fresh water) after 
which it is seasoned with a little salt. In this soup the fat is in the form of a 
very fine emulsion and the taste is so pleasant that a more nearly normal stimu- 
lus to gastric secretion is offered. The patient is now allowed to take 300 c.c. 
of this soup, while the remaining 50 c.c. are retained for a determination of the 
fat content. The contents are withdrawn, one hour after the meal, from the 
stomach which must have been thoroughly washed out prior to the administra- 
tion of the meal. 1 

We then determine the absolute amount of fat remaining in the stomach 
after the test digestion and compare this amount with that introduced. As 
we cannot be sure that the entire stomach contents have been withdrawn, we 
must know the residual amount of gastric juice. For this purpose one resorts 
to the method of Mathieu, which will be discussed later (p. 58). 

The amount of fat both in the original soup and in the withdrawn stomach 
contents is then determined and the total gastric juice calculated. This method 
of fat determination will be given in detail under Milk, to which the reader 
is referred. 

Calculation of Results. 

"The following calculations are possible from a consideration of the residue 
from the acidity of the gastric nitrate, and from the difference between the 
amount of fat found in the ingested flour soup and that found in the expressed 
contents. 

" By the addition of the value X, found in the calculation of the residue 
(P« 5^)> to the amount of contents expressed after one hour, there is obtained 
the volume of the contents which were actually present in the stomach at the end 
of that period. This we designate as To. From the absolute fat-content of 
To, there can be determined how much of the volume can be ascribed to the 

1 Sahli (Lehrbuch d. klin. Untersuchungsmethoden 6 Aufl., 1913, I, 656) apparently 
now prefers a soup prepared from Maggi or Teston bouillon and yolk of egg instead of the 
above. 



GASTRIC CONTENTS. 57 

ingested flour soup. The amount of fat remaining in the stomach serves, there- 
fore, as a measure for the amount of soup remaining. This is designated as 
Su. Representing this mathematically, we have the proportion To: Su: : F: f, 
in which F represents the percentage fat content of the soup and f that of the 
expressed contents. To — Su will give, of course, the volume of gastric juice 
in the expressed contents. If the acid-content of To has been determined, it is 
possible from these data to proceed further and to calculate what acidity was 
possessed by the pure gastric juice as it was excreted. Thus, if 75 c.c. of pure 
gastric juice are present in the stomach contents, whose volume amounts to 
150 c.c. with 2 per cent, acidity, then the acid-content of the pure gastric 
juice is evidently 4 per cent. The determination of this acidity may be ex- 
pressed by the proportion, To: A: : Ma: a, in which To represents the amount of 
expressed contents including the residue, Ma the amount of secretion contained 
in the expressed contents (To — Su), A equals the acidity of the pure secretion 
in per cent, and a the percentage acidity of the expressed contents" (Sahli). 
It is important to remember in selecting a test meal for any given case 
that the tastes of no two persons are alike and that no two persons will react 
identically toward the stimulation of the same test meal. The results can only 
be comparative and have, in this sense, some value. Too much rigidity in 
administration of such meals will lead frequently to mistakes in diagnosis, so 
that one should learn to vary his test meals rather than to rely upon a single one 
in all cases. Another point to be borne in mind is that the meals should be 
removed at the time of optimum secretion, which may not in all cases be at the 
end of one hour with the Ewald meal. 1 "The technic commonly employed for 
estimating gastric function is entirely inadequate, inasmuch as it indicates 
but one phase in a constantly changing cycle, and that phase is by no means 
always the high point in the digestive curve" (Rehfuss, Bergeim and Hawk). 

III. Macroscopic Examination. 

The gastric juice is a clear, colorless, easily filtered, levorotatory fluid 
having a distinctly acid reaction, an acid taste, and a characteristic odor. Its 
specific gravity, when the stomach is empty, ranges between 1004 and 1006.5; 
after the ingestion of food from 1010 to 1020 and more than 1020 when the 
production of acid is diminished (Landois). Its cryoscopic point is —0.38 
degree to —0.444 (Roth and Strauss). 

Amount. 

The figures for the total amount of gastric juice secreted in 24 hours are 
variable. Calson 2 shows that an adult secretes a total of about 1500 c.c. in 24 
hours, appetite being a potent factor in this process. The amount of fluid 
obtained one hour after an Ewald meal, is from 20 to 50 c.c, although larger 
amounts ranging from 200 to 500 c.c. indicate either diminished motility or 
hypersecretion, on the one hand, or dilatation associated with pyloric stenosis, 
on the other. It is to be remembered that the quantity of juice secreted is in- 

1 See Ettinger, Internat. Beitr. z. Ernahrungsstor., 1913, IV, 454. 

2 Am. Jour. Physiol., 1915, XXXVII, 50. 



58 DIAGNOSTIC METHODS. 

fluenced by the appetite and by the amount and character of the food taken, as 
well as by the age and sex of the patient and the time of day at which the food 
is taken. The largest amounts of gastric juice are found in cases of hyper- 
secretion when it is not uncommon to find a liter or more of gastric juice in the 
non-digesting stomach. 

In order to determine the total amount of gastric juice secreted, one cannot 
rely upon the quantity removed as there is always a slight residuum. The 
method of Mathieu and Remond is commonly used for such determinations. 
This gives results very nearly exact, at least for clinical purposes. With this 
method the gastric contents are removed, as nearly as possible, after an Ewald 
meal. A definite quantity of water, usually 300 c.c, is then poured into the 
stomach through the tube and is thoroughly mixed by moving the funnel up 
and down and by pressure upon the stomach. As much as possible of this 
added fluid and the remaining gastric juice is removed and collected in a sepa- 
rate vessel. The acidity of the undiluted as well as of the diluted stomach con- 
tents is then determined by titration. From the difference in these two values 
conclusions may be drawn as to the degree of dilution and to the residual 
amount of stomach contents which was not expressed. The amount expressed 
plus the residual amount equals the total gastric contents. 

The following is the method of calculation according to Mathieu: 

Let a = acidity of the undiluted gastric contents. 

Let b = acidity of the diluted gastric contents. 

Let x = amount of the test meal remaining in the stomach after 
expression. 

Let 300 c.c. = the amount of water introduced into the stomach for dilu- 
tion. Then 

a : b : : x + 300 : x 
ax = b (x + 300) 
300 b 



x = 



a — b 



An absolutely accurate result, in the study of gastric activity, can be ob- 
tained only when the total quantity of gastric juice is known. 1 It is, therefore, 
necessary in stating, for instance, the acidity of a stomach contents to calculate 
the total available acidity rather than the mere degree of acidity. It is self- 
evident that a stomach contents expressed in the ordinary way, which shows an 
acidity of 40 , may have this acidity in a total quantity of 50 c.c, while an acidity 
of 40 , with a total quantity of 200 c.c, would represent actually four times as 
much hydrochloric acid available. It would seem, therefore, to the writer 
that the method of representing acidity in terms of degrees without any refer- 
ence to the amount of gastric contents is absolutely irrational. 

Color. 

Gastric juice is normally a practically colorless liquid, although at times 
it may be somewhat opaque and, therefore, much whiter in color. Variations 

1 See Rehfuss, Jour. Am. Med. Assn., 1915, LXTV, 569. 



GASTRIC CONTENTS. 59 

in this colorless fluid are observed after test meals due to admixture of various 
food products, so that we may have distinctly brownish colorations due to the 
tea or particles of toasted bread, while in the test meals consisting of meat the 
color may be more of a reddish tone. 

Pathologically, we may find a distinct red color due to the presence of blood. 
This bright red color comes from the presence of fresh blood from a hemor- 
rhagic gastric ulcer or may be derived from abrasions of other portions of the 
alimentary tract. If the blood has been thoroughly mixed with the stomach 
contents for some time it may appear in the form of a brownish-black deposit, 
the so-called coffee-ground material. The blood in these cases is in the form 
of hematin and must be tested for as later outlined. This coffee-ground 
appearance is particularly evident in cases of gastric carcinoma. 

The color of the gastric contents may be either a yellow or a green, due to 
the presence of bilirubin in the former case and biliverdin in the latter. This 
biliary pigment should be detected by the tests outlined under Urine. The 
presence of bile in the gastric contents is indicative of duodenal occlusion. 

In cases of intestinal occlusion below the duodenum we occasionally find 
fecal matter in the gastric contents. This is characterized by the brownish- 
black coloration and by its intense odor. 

Odor. 

The normal gastric juice is practically odorless or very slightly sour. Ad- 
mixtures of material coming from the intestines cause a very intense odor, 
while the material rising from abscesses along the alimentary tract above the 
stomach will frequently give rise to a very offensive odor. In the vomitus ob- 
tained under various pathological conditions the odor may be very characteristic. 
Thus, in uremia we may find a distinct odor of ammonia, in alcohol intoxication 
a distinct alcoholic odor is evident, while in cases of stagnation of gastric con- 
tents ail intensely strong odor is observed. In cases of dilatation we frequently 
find the organic acids so much increased in amount that distinct odors are 
noticeable. 

Consistency. 

The normal stomach contents are usually watery in character, but may 
vary due to admixture with extraneous material. After test meals or following a 
vomiting spell we may find portions of unchanged protein or carbohydrate 
material. The amount of bread taken with an Ewald meal should be so far 
digested in one hour as to form a puree-like mass which settles out on standing. 
Various food residues are, of course, present in the vomitus so that the consist- 
ency and appearance may give us much valuable information regarding the 
digestive process. In cases of mucous catarrh or in those showing either a 
diminution or an increase in the amount of hydrochloric acid we may find after 
a test meal the presence of large amounts of tough, slimy, mucoid material, 
which may be so abundant as practically to make it impossible to filter the con- 
tents. The consistency of such material may be almost that of a paste, or may 
be simply that of a thick syrup which on pouring from the vessel onto the filter 
will form distinct mucoid threads. The presence of an increased amount of 



60 DIAGNOSTIC METHODS. 

mucus is of some diagnostic importance and should, therefore, be looked for 
under all circumstances. 

Gastric Contents from Fasting Stomach. 

The stomach is practically never empty, always containing a certain 
amount of acid fluid. 1 Boas considers anything between ioand iooc.c. as a nor- 
mal amount of material for the fasting stomach. Anything above this amount 
would mean either motor insufficiency or hypersecretion. One may differen- 
tiate these two conditions by washing out the stomach at night, when the mate- 
rial withdrawn in the morning will be extremely scanty if the condition is one 
of motor insufficiency. 

This fluid from the fasting stomach is thin, has a specific gravity of 1004 
to 1005, contains some free hydrochloric acid, no lactic acid, and no bacteria. 
It is very commonly bile-stained, may be alkaline from the presence of pan- 
creatic juice and may contain large amounts of mucus. As such material is 
always found in the fasting stomach it is well to make it a rule to wash out the 
stomach the night before giving a test meal. 

Vomitus. 

In those cases which are associated with frequent vomiting we may obtain 
much valuable information from the examination of the ejected material. 
The amount of material vomited will depend, of course, upon the motility of the 
stomach. In cases of dilatation or of stenosis we frequendy find two or three 
quarts of material, while in conditions associated with hypermotility we may 
have simply a scanty highly mucoid vomitus. The presence of food particles 
will give much information as to the digestive power of the stomach. If 
undigested meat fibers are found in the vomitus, ejected three hours after eating, 
one may assume more or less disturbance of protein digestion. If particles of 
unchanged bread are found, three hours after taking, the disturbance in protein 
digestion is probably more marked than in the case of the meat fibers. If an 
individual vomits bits of food more than seven hours after a meal, some im- 
pairment of motility must exist, according to Sahli, for after that interval even a 
hearty meal should have completely left the stomach. The vomiting of an acid 
liquid containing no food particles is quite characteristic of hypersecretion of 
gastric juice. 

The degree of acidity of the vomitus as well as the amount of hydrochloric 
acid present very frequently enables us to judge of the activity of the juice. 
These figures will not be as reliable as are the ones obtained after a test meal, 
but may serve in cases in which the stomach-tube cannot be passed. 

Frequently one finds a vomitus which is quite foamy and smells strongly 
of the volatile fatty acids. In such conditions we may assume a diminution in 
the amount of hydrochloric acid, which normally prevents the occurrence of 
any such decomposition, or we may ascribe this condition to simple stagna- 
tion of the gastric contents. Such contents will show microscopically the 

1 See Carlson, Am. Jour. Physiol., 1915, XXXYTI, 50; also, Rehfuss, Bergeim and Kawk, 
Jour. Am. Med. Assn., 1914, LXIII, 11 and 909; Fowler, Rehfuss and Hawk, Ibid., 1915, 
LXV, 102 1. 



GASTRIC CONTENTS. 6l 

presence of large numbers of sarcinae ventriculi, yeast fungi, and various 
bacteria. 

The blood in the vomitus varies from a slight streaking of the material to a 
fluid which shows intimate mixing with the gastric contents. In cases of re- 
cent hemorrhage, which is particularly common in ulcer of the stomach, an 
abundant admixture of fresh arterial blood or of dark coagulated blood is 
observed. Brown or black coffee-ground-like material is particularly 
suggestive of carcinoma, although the same condition may result from 
erosion of the gastric mucous membrane when associated with hyperacidity or 
hypersecretion. 

An admixture of bile, producing a yellowish or greenish discoloration, 
may occur with any type of vomiting, but more especially from an empty stom- 
ach and in that associated with duodenal obstruction. A biliary vomiting 
is frequently observed in peritonitis and may be due to the fact that there is no 
counterpressure from the gastric contents to prevent regurgitation from the 
duodenum. This green vomitus may not always be due to the presence of 
bile, but may come from contamination with various chlorophyll-containing 
organisms. 

Almost all types of vomitus contain mucus. In some cases we find abun- 
dant tough, slimy masses which seem to be indicative of mucous catarrh of 
the stomach or of a diminution in the amount of hydrochloric acid. 

Fecal vomiting is a sign either of complete motor insufficiency of the intes- 
tine as found at times in peritonitis, or indicates intestinal obstruction, either 
in the lower part of the small intestine or in the large bowel. The brownish- 
black color of this vomitus and the distinct odor render it very characteristic. 

Asiatic cholera and cholera nostras are associated with a vomitus which 
is abundant, alkaline in reaction, contains white flakes of mucus and epithelial 
cells, and large numbers of bacteria, both Koch's spirillum and the Finkler- 
Prior spirillum and various other unidentified types. This vomitus of cholera 
is known as the "rice-water" vomitus. 

The time at which vomiting occurs is frequently of great importance from 
the diagnostic standpoint. If it be at the height of digestion and during in- 
tense pain the condition is probably one of ulcer. If during or shortly after 
eating we may have either gastritis, a neurosis, or cancer. If it is frequent in 
the morning before breakfast and seems to be independent of eating the con- 
dition is probably one of dilatation. While these statements are not infallible, 
yet they are applicable in the majority of cases. 

Gastric Contents After Test Meals. 

The amount of material obtained after a test meal has some diagnostic 

importance. As previously stated, one obtains after an Ewald or Boas meal 

from 20 to 50 c.c, of contents, but these figures may vary to as high as 500 c.c. 

Hypersecretion or motor insufficiency are the chief causes of such increased 

amounts, the former being more probable if a large amount of free hydrochloric 

acid is present along with the excessive amount of fluid. x A larger proportion 

1 van Spanje (Nederl. Tijdschr. v. Geneesk., 1913, LVIII, 213) calls attention to the dry 
test meal of triscuit in estimating the motor efficiency of the stomach. 



62 DIAGNOSTIC METHODS. 

of solid undigested material is observed in cases of pure motor insufficiency, 
but we frequently have a combination of both conditions. Absolute proof of 
the diminished motility is found in the presence of more than a trace of food 
in the stomach seven to eight hours after a meal. The general appearances 
of the material obtained after a test meal will be practically those previously 
discussed. 

IV. Microscopic Examination. 

The microscopical examination of the gastric contents is usually made on 
material withdrawn from the stomach after test meals, but the vomitus is oc- 
casionally examined. The gastric juice is practically never free from remnants 
of food, such as meat threads or starch granules, although nothing has been 
taken for many hours. Moreover, small masses of mucus, which occasioDally 
assume a snail-like spiral form, and saliva which is recognized by the presence of 
large flat epithelial cells and the so-called salivary corpuscles are quite frequent. 
A few bacilli and yeast cells are almost always observed. 

As such elements as the ones above mentioned are present in all gastric 
juice, we must not attach undue importance to the presence of small amounts 
of such material in the contents obtained after a test meal. After the Ewald 
meal one rarely finds anything beyond the presence of numerous starch granules 
and more or less mucoid material, along with bacteria of the various types which 
flourish particularly in the buccal and gastric cavities. 1 

In cases associated with diminished motility of the stomach we may find 
remains of food which has been introduced many hours previously. In such 
specimens we observe numerous fat globules or fatty acid crystals, many vege- 
table fibers and cells and a few red blood-cells which have come from slight 
abrasion of the mucous membrane of the pharynx by the stomach-tube. These 
red cells usually are much altered in appearance by the hydrochloric acid and 
do not show their ordinary hemoglobin color, but take on a more brownish tint, 
which is due to the presence of hematin. 

Boas-Oppler Bacillus. 

This organism is found quite commonly in patients suffering with carci- 
noma of the stomach, and is almost always absent in nonmalignant disease. 
It is found more frequently in the gastric contents at a time when lactic acid 
is present in large amounts, so that in the incipient stages of carcinoma these 
organisms may be absent. These bacilli are very long (3 to 10 microns), 
1 micron broad, and are frequently joined end to end forming very long chains. 
They are readily stained with the usual aniline dyes and by Gram's method and, 
on treatment with iodin, take on a brown color which distinguishes them from 
the large mouth bacillus (leptothrix buccalis), which stains blue with iodin. 
This organism appears to be identical with the bacillus bulgaricus, one of the 
lactic acid group, and is not an organism sui generis* It is not absolutely 
pathognomonic of carcinoma of the stomach, although Smithies 3 demonstrates 



1 See Lyon, Am. Jour. Med. Sc, 1915, CL, 402. 

2 Gait and lies, Jour. Path, and Bacterid., 1914, XJX, 239. 

3 Jour. Am. Med. Assn., 1913, LXJ, 1793. 






GASTRIC CONTENTS. 63 

its presence in 93.8 per cent, of 566 cases of pathologically verified carcinoma. 
Owing to the absence of hydrochloric acid in such cases, this organism develops 
and produces the lactic acid found. 
Sarcinae. 

Occasionally in normal gastric juice and especially in cases of dilatation 
with marked fermentation one finds the so-called sarcina ventriculi which 
are cocci arranged in squares or tetrahedra which resemble, very much, cotton 
bales. These organisms have no pathologic significance, but are indicative 
of stagnation of gastric contents. Along with these sarcinae one may find large 
numbers of yeast cells. 




Fig. 16 — Boas-Oppler bacilli. (Hemmeter.) 

Protozoa. 

These unicellular parasites have been occasionally found in the gastric 
contents. Flagellates, amebas, and monads seem to be more frequent than the 
other types of protozoa. 1 They seem to be more commonly found in cases of 
carcinoma of the stomach. 
Fragments of Tissue. 

Frequently small shreds of mucous membrane are found in the expressed 
gastric contents. One finds these in cases of chronic gastritis, ulcer, hyper- 
chlorhydria, and especially in cancer. These tissue fragments should be studied 
carefully under the microscope, as not infrequently a diagnosis of cancer is pos- 
sible from such examination. 
Crystals. 

Various types of crystal are occasionally noted in the gastric contents, 
among which may be mentioned bile acids, cholesterin, fatty acids, leucin, 
tyrosin, and calcium oxalate. If the reaction of the juice is alkaline triple 
phosphate crystals may appear. 

V. Chemical Examination. 

The chemical examination of the gastric juice is the most important of 
all laboratory methods in the diagnosis of various pathologic gastric conditions. 
1 See Smithies, Am. Jour. Med. Sc, 1912, CXLTV, 82. 



64 DIAGNOSTIC METHODS. 

As previously stated, the acidity of gastric juice is referable to the presence of 
free and combined acids. The free acidity is traceable largely to hydrochloric 
acid, although organic acids, such as lactic, acetic, and butyric, may increase 
the free acidity under abnormal conditions. Besides this free acidity, we have 
hydrochloric acid which is bound chemically to the protein substances and does 
not react with tests for free acidity. There are also present in the gastric juice 
acid salts, especially the sodium dihydrogen phosphate (NaH 2 P0 4 ). 

Besides these factors which have to do with the reaction of the gastric 
juice, we find certain ferments which act only in the presence of the free hy- 
drochloric acid. 1 The first of these, pepsin, has the power of acting upon al- 
bumin in an acid medium and converting it, through various stages, into lower 
splitting products of albumin. This peptic digestion will be discussed in 
detail later. A second ferment, known as rennin, lab, or chymosin, has the 
power of curdling milk by coagulating the casein. A third ferment, lipase, 
acts upon fat, especially when this is present in a finely divided form. This 
lipolytic action is not ordinarily great, but should nevertheless be remembered. 
The experiments of Sahli show that this action is negligible during the period 
covered by the administration of his test meal. 

Although many statements have been made to the contrary, the gastric 
juice, through the agency of the hydrogen ions of its free hydrochloric acid, 
acts upon certain polysaccharides, especially cane sugar, hydrolyzing them into 
the simpler monosaccharides. Careful determinations indicate that the speed 
of inversion is about the same as that of an equal strength of hydrochloric 
acid, so that we do not need to assume any ferment action. 

Besides these substances gastric juice contains a small amount of albumin, 
carbohydrates, and various inorganic salts. None of these constituents have 
any importance from a clinical standpoint and will be disregarded. 

(i). Total Acidity. 

As previously stated, the total acidity of the gastric juice is referable to the 
presence of free and combined hydrochloric acid, organic acids, and acid salts. 
This factor may be readily determined by titrating 10 c.c. of gastric juice 
with tenth-normal sodium hydrate, using phenol phthalein as an indicator. 
This indicator is colorless in the presence of acid and becomes red at the point 
of neutralization, being used as a i per cent, alcoholic solution. On adding a 
few drops of this solution to the unfiltered gastric contents, a white cloud will be 
observed due to the precipitation of the reagent by the water of the gastric juice. 
The titration is carried to the point at which the addition of sodium hydrate 
produces a definite pink color which remains permanent and does not deepen 
on the addition of further alkali. If sodium chlorid be added to the point of 
saturation of the gastric contents, the end point becomes somewhat sharper 
owing to the diminished dissociation which the disodium hydrogen phosphate 
undergoes into sodium dihydrogen phosphate in the presence of increased 
sodium ions. This precaution is rarely taken, however, as the clinical result 
is never so accurately determined as is the scientific factor. 

1 See Ewald, Deutsch. Arch. f. klin. Med., 1912, CVT, 498. 



GASTRIC CONTENTS. 65 

The total acidity varies between rather wide limits. Normally it ranges 
from 75 to ioo°, being made up of approximately 5o°of free hydrochloric acid, 
25 of combined hydrochloric acid, and 25 of organic acids and acid salts. 
The chief variation under normal conditions is an increase in the combined 
hydrochloric acid and a decrease in the organic acids and acid salts. 

In pathologic conditions we may find the total acidity high with very 
little free hydrochloric acid, or we may find the total acidity low, with a normal 
amount of hydrochloric acid present. 

Sahli has stated the variations in the acid factors of the stomach contents 
as follows: If the total acidity is high and the hydrochloric acid is normal, 
the high acidity can be due only to a deficient motility and absorption and hence 
we find an increase in the organic acids. Such a gastric juice may show 
lactic acid, but will more probably give the tests for the other organic acids. 
A low total acidity with an excess of hydrochloric acid shows that the motility 
and absorptive powers of the stomach are good. If the total acidity be mod- 
erate and free acid small in amount, a poor motility may be assumed. Gen- 
erally speaking, when much lactic acid is present we find low HC1 and only 
combined HC1; that is, diminished secretion and diminished motility. No 
lactic acid is found when the HC1 is normal or increased. 

(2). Free Hydrochloric Acid. 

A number of tests have been devised for the detection of free hydrochloric 
acid in the stomach and its differentiation from lactic and acetic acids. It 
should be remembered that the tests outlined below are not specific tests 
for hydrochloric acid, but are common to all mineral acids. Many of these tests 
react also with the organic acids, providing they are present in sufficient con- 
centration. The efficiency of any acid is due merely to the ionic decomposition 
which it suffers when in solution; in other words, is due to the presence of free 
hydrogen ions. Hydrochloric acid appears to be more efficient in the digestive 
processes than do the organic acids, owing simply to its greater degree of disso- 
ciation. The tests commonly employed in clinical work are based upon the 
reaction which certain coloring matters undergo when treated with free hydro- 
chloric acid. These tests can be, therefore, only approximate and must be used 
with discretion in scientific work, although in clinical work they are near 
enough for all purposes. Were we able to express completely the stomach con- 
tents and thus to obtain material which would give us absolute data, we would 
then require better clinical methods. What should be measured in testing 
stomach contents for free acidity is the number of hydrogen ions which the 
gastric juice contains, since this is the important factor in the efficiency of the 
juice. 1 This may be determined directly by measuring the speed of any cata- 
lytic reaction due to hydrogen ions, the most convenient one being the rate of 
inversion of cane sugar. 

It is wise to have some quick method by which one may determine the 
presence of a free acid in the gastric juice. This may be done with litmus 
which will, however, not show the presence of free hydrochloric acid to the ex- 

1 See Michaelis, Die Wasserstoffionen Konzentration, 19 14, Springer, Berlin. 



66 DIAGNOSTIC METHODS. 

elusion of other acids or acid salts. To determine whether the acid reaction 
is due to free acid it is customary to employ the Congo-red paper. This con- 
sists simply of filter-paper which has been saturated with an alcoholic solution 
of Congo-red and dried. On treating gastric juice with such paper we obtain 
a blue color in the presence of free acid. 1 This paper reacts with a blue color 
to any free acid so that one should never assume the presence of free hydro- 
chloric acid when he obtains a blue coloration. Although many writers state 
that a blue coloration is never given by free organic acids, the writer has seen 
too many cases in which distinct action was referable either to free lactic or 
free acetic acids to agree with this statement. The tests which are applicable 
to the detection of free mineral acids in general may be used as indicative of 
free hydrochloric acid, as this is the only mineral acid which one would ordinarily 
find in the gastric contents. 

Qualitative Tests. 

(a). Tbpfer's Test. 

The test is based upon the coloration which a 0.5 per cent, alcoholic solu- 
tion of dimethyl-amido-azobenzol takes when treated with gastric juice contain- 
ing free hydrochloric acid. A few c.c. of unliltered gastric juice are placed in a 
dish and one or two drops of the above solution added. In the presence of 
free mineral acids a carmin red color is obtained. This reagent is a very deli- 
cate one and does not react to organic acids unless they are present in amounts 
exceeding 0.5 per cent. The coloration with free hydrochloric acid varies in 
intensity with the amount of acid present, and may range from a deep orange 
to an intense carmin. According to Simon, lactic acid does not give the typical 
red color with this reagent, especially if albumoses are present, unless it be in a 
concentration of at least 1 per cent. This reagent will detect the presence of 
0.02 parts of hydrochloric acid per thousand. 

(b). Gunzburg's Test. 

The reagent employed in this test consists of 2 grams of phloroglucin and 1 
gram of vanillin dissolved in 30 c.c. of absolute alcohol. This yellowish solu- 
tion should be kept in dark bottles, as it gradually changes to a dark red and then 
to brown when exposed to the light. Boas claims that the reagent becomes more 
delicate and stable if one dissolves the phloroglucin and vanillin in 100 c.c. of 
80 per cent, alcohol. 

Two or three drops of this solution are added to an equal amount of the 

gastric juice contained in a porcelain dish and the mixture evaporated over 

water-bath. In the presence of free mineral acid a rose-red color is developed, 

varying in intensity with the amount of acid present. This mixture must not 

be boiled or heated too rapidly as the resulting color will then be brown or 

1 Holmgren (Deutsch. med. Wchnschr., 19 11, XXXVII, 247) has devised a quantitative, 
as well as qualitative, test for free HC1 using the capillarity of such Congo-red paper as a basis 
of estimating the quantity of HC1, the discolored zone varying in width with the degree of acidity. 
This method of " capillary analysis" is, however, not as constant and reliable for quantitative 
purposes as are the titration methods, as Mattisson (Arch. f. Verdauungskr., 1913, XIX. 79 
and 226) and Schmidt (Biochem. Jour., 1913, VII, 231) have shown. Aberg (Hygiea, LXXYI, 
105) advises the use of tincture of cochineal or lacmoid as an indicator in this test. See, also, 
Sochanski, Arch. f. Verdauungskr., 19 14, XX, 317. 



GASTRIC CONTENTS. 67 

brownish-red and may mislead one into believing that no free hydrochloric acid 
is present. 

This test does not react to organic acids or to acid salts, nor is it interfered 
with by the presence of products of food digestion. It may, therefore, be used 
with the unfiltered gastric juice. This test reacts with the rose-red color in the 
presence of 0.05 parts of HC1 per thousand. 1 

(c). Boas' Test. 

This reagent consists of 5 grams of resorcin and 3 grams of cane sugar 
dissolved in 100 grams of 95 per cent, alcohol. It has the same delicacy as 
Giinzburg's test and is more stable. The test is applied in the same way as 
the preceding, taking particular care to use a low flame in evaporating, and 
gives a rose-red or vermilion color in the presence of mineral acids. This color 
gradually fades on cooling and is not given by organic acids or acid salts. 

(d). Tropeolin Test. 

The reagent for this test is a saturated alcoholic solution of tropeolin 00. 
This test is applied in the same way as the preceding and gives a lilac-blue color 
in the presence of free acid. 2 This test is not as delicate as the preceding, re- 
acting only in the presence of 0.3 parts of free hydrochloric acid per thousand, 
and has the objection that it strikes the blue color much more easily with the 
organic acids. 

Other tests have been advocated for the qualitative detection of free 
hydrochloric acid, but they are not as delicate as the above and have nothing 
to justify their existence. 3 Of the tests given, the Giinzburg test seems to be 
the most reliable, although the Topfer's test is clinically sufficient and has the 
advantage of being much less expensive and more convenient than is the for- 
mer reagent. 

Quantitative Methods. 

The quantitative estimation of free hydrochloric acid is of great importance 
in the study of all pathologic conditions of the stomach. Any determination 
made with our present methods must have reference to the fact that the acidity 
of the gastric juice is due almost entirely to the free hydrochloric acid. While 
this is not absolutely true, yet the organic acids are rarely present in sufficient 
amounts to react with the indicators in conditions in which hydrochloric acid 
is normal or increased in amount. However, in the conditions associated with 
diminished amount of hydrochloric acid one must be on his guard in the inter- 
pretation either of the qualitative or quantitative tests for free hydrochloric acid. 
The writer has seen several cases in which all of the indicators, with the excep- 
tion of Giinzburg's reagent, showed positive results for hydrochloric acid, the 
cases being those in which no free hydrochloric acid was actually present. Bear- 
ing this in mind we may determine the acidity, referable to free hydrochloric 
acid, by the titration of a known amount of the unfiltered gastric juice with 

1 See Christiansen, Biochem. Ztschr., 1912, XLVI, 24, 50 and 71. 

2 Fittipaldi (Gazz. d. osp., 1913, XXXIV, 289) uses this reagent for quantitative purposes. 

3 See, however, Cipallina, Riforma Med., 1913, XXIX, 505. 



68 DIAGNOSTIC METHODS. 

tenth-normal sodium hydrate solution, using as indicators the solutions men- 
tioned under the head of qualitative tests. * 

Mintz Method. 

As the Giinzburg reagent is the most delicate and reliable of all the tests 
for free hydrochloric acid, it is wise to use this reagent as an indicator. The 
test as usually followed by the writer is to add 20 to 30 drops of the Giinzburg 
reagent to 10 c.c. of the gastric juice. On adding the sodium hydrate solution 
no color change will be visible, as the reaction takes place only when the solu- 
tion is warmed. This warming cannot be done directly as the evaporation 
would necessarily have to proceed to the point at which loss of hydrochloric acid 
might occur. Following the recommendation of Sahli, the rod with which 
the solutions are stirred is warmed before being used. As the neutralization 
point is reached the red color, originally noted on the sides of the warm rod, 
disappears. 

Topfer's Method. 

This method is the simplest, is the most generally used, and at the same 
time is one of the most delicate of all the clinical quantitative methods for 
free hydrochloric acid. It consists in the use of dimethyl-amido-azobenzol as 
an indicator, the titration of the unfiltered gastric juice being done with tenth- 
normal sodium hydrate solution. Although this reagent does give, under some 
conditions, a red color in the absence of free hydrochloric acid when the organic 
acids are largely increased, yet such conditions are so rarely found in clinical 
work that the result of test-tube experiments cannot be applied to clinical cases. 
The experiments of Simon show that lactic acid must be present to the ex- 
tent of 1 per cent, before any cherry-red color is obtained, providing albu- 
moses are present. As these latter substances are always found in the 
gastric juice after intake of protein material, one can readily see that lactic 
acid need not be considered. We find, however, conditions associated with 
fermentative processes in the stomach in which acetic and butyric acids are 
present in fairly large amounts. These acids will give a red color with the indi- 
cator, but should not mislead as their strong odor in such concentrations 
permits of easy recognition. 

In the titration the sodium hydrate solution is added from a buret to the 
unaltered gastric juice to which one or two drops of indicator are added for 
every 10 c.c. of juice. In the presence of free hydrochloric acid this indicator 
strikes a distinct cherry-red tone and thus enables the worker to decide at 
once as to the presence or absence of the acid. No evaporation is necessary, 
hence the test has the advantage of simplicity and does not occasion any loss 
of substance. As the sodium hydrate solution is added the reddish tint of 
the mixture changes to a distinct yellow. The titration must be carried to the 
point at which every trace of red disappears and the color becomes a pure 
yellow. This reaction requires considerable experience, hence the writer would 
advise the student to make his titration with a known solution of hydro- 
chloric acid so that he may become familiar with the end point. 

1 See Knapp, New York Med. Jour., 1913, XCVII, 437. 



GASTRIC CONTENTS. 69 

Although the writer formerly used the filtered contents in his estimations, he 
finds that filtration causes distinct changes in the composition of the fluid, owing 
to adsorption of free hydrochloric acid. He advises, therefore, the use of the 
thoroughly mixed unfiltered contents, unless there is a very large amount of 
mucus present. Under this latter condition one may use the filtered contents. 
If one wishes to clarify the fluid for the determination of pepsin, it is wise to 
centrifuge the specimen instead of filtering. * 

Amount of Free Hydrochloric Acid. 

Although the general idea prevails that the normal human gastric juice 
contains free hydrochloric acid to the extent of 0.2 to 0.3 per cent., recent studies, 
under properly controlled conditions, on both lower animals and man, indicate 
that this amount reaches a constant figure more nearly that of 0.5 per cent, in 
cases in which the freshly secreted normal gastric juice is examined. Carlson 2 
calls attention to the fact that normal human gastric juice secreted at a fairly 
high rate is equal in total acidity to the acidity reported by clinicians for so-called 
hyperacidity, and states that "there is no evidence that the gastric glands under 
any pathologic conditions are able to or do secrete a juice of higher than normal 
acidity." Although it is impossible to deny the existence of cases of hyper- 
acidity, these experiments and conclusions are, to a certain extent, in conformity 
with clinical observations, as we unquestionably observe many cases of high 
acidity with no symptoms of this condition and, on the other hand, symptoms of 
hyperacidity in cases of real hypoacidity. Hence, we are forced to the conclu- 
sion that the same amount of acid may indicate a hyperacidity in one case and 
not in another. The fallacy of relying on percentage relations instead of relating 
the symptoms of hyperacidity to a hypersensitiveness of the gastric mucosa toward 
certain degrees of hydrochloric acid is, therefore, evident. 

This normal acidity does not remain in the digesting stomach at this higher 
level for a great length of time, being relatively soon lowered to a level of opti- 
mum acidity of about 0.2 to 0.3 per cent., according to Boldyreff, 3 by neutraliza- 
tion by the mucus secreted by the gastric glands, by the combination of the acid 
with the protein of the food and, especially, by reflux of alkaline juices from the 
duodenum into the stomach. At the height of digestion we find, therefore, that 
the acidity of normal gastric juice may range from 0.2 to 0.5 per cent., this 
optimum varying in different subjects and with the type of food in the stomach. 
Under ordinary conditions this excess of free hydrochloric acid should be evident 
in 45 to 60 minutes after an Ewald breakfast, but this time varies with the 
individual case. It is evident, therefore, that a diagnosis of hyperacidity or 
hypoacidity cannot be absolutely made by reference simply to the chemical 
analysis of a gastric contents. 4 

Certain clinical conditions lead to an excretion of gastric juice which is 
normal, increased, or diminished in amount of free hydrochloric acid. We 

1 See Christiansen, Biochem. Ztschr., 1912, XLYI, 82; Zunz, Handb. der Biochem. Ar- 
beitsmeth., 1914, VIII, 44; Seidl, Arch. f. Verdauungskr., 1915, XXI, 196. 

2 Am. Jour. Physiol., 1915, XXXVIII, 248; also, Menten, Jour. Biol. Chem., 1915, XXII, 

34i. 

3 Quart. Jour. Exper. Physiol., 1914, VIII, 1. 

4 See Rehfuss and Hawk, Jour. Am. Med. Assn., 1914, LXIII, 20S8. 



70 DIAGNOSTIC METHODS. 

must, therefore, have a method of interpretation of the free HC1 acidity of the 
stomach. It is customary to report the results of titration of a given specimen 
of gastric juice in one of two ways: 

(i). We may represent the acidity referable to free hydrochloric acid by 
the number of c.c. of tenth-normal sodium hydrate necessary to neutralize ioo 
c.c. of unfiltered gastric juice, using dimethyl-amido-azobenzol as an indicator. 
This is called degree or percentage of acidity. Thus, if 5 c.c. of tenth-normal 
sodium hydrate were used to neutralize 10 c.c. of unfiltered gastric juice, the 
degree or percentage of acidity would be, obviously, 50. 

(2). We may report the actual amount of free hydrochloric acid present. 
This is the more scientific way, as we have a much better means of comparison 
with the normal standards. One c.c. of tenth-normal sodium hydrate neutral- 
izes 0.00365 gram of free HC1. If, now, we multiply this factor by the number 
of c.c. necessary to neutralize iooc.c.of unfiltered gastric juice, we obtain a figure 
representing the absolute amount of free HC1 in the gastric content. Thus, 
if the gastric juice showed an acidity of 50 degrees, we would have 0.1825 
(50 x 0.00365) gram of HC1. The usual text-book statement is that the free 
hydrochloric acid is normally about 40 degrees, but the writer is accustomed to 
consider an acidity of 50 to 55 as much more nearly normal than one of 40. 
Euchlorhydria. 

This is a condition in which the amount of free hydrochloric acid is between 
0.2 and 0.3 per cent. In speaking of percentage one must not confuse the 
two types of percentage reckoning. Thus, 10 per cent, or io° of HC1 rep- 
resents only 0.0365 gram or true per cent, of hydrochloric acid. The lower 
figure of 0.2 per cent, is given for euchlorhydria owing to the fact that normal 
variations may permit of this low point, although the higher figure of 0.3 per cent, 
is the more usual. When this euchlorhydria exists in the presence of clinical symp- 
toms pointing to gastric disturbance, we usually have to do with a neurosis. Gas- 
tritis may be absolutely ruled out, a carcinoma excluded except when the new 
growth has taken place upon an old ulcer and an ulcer practically always ruled out, 
although this latter may show an euchlorhydria. This condition may be asso- 
ciated with a certain amount of atony along with more or less marked dilatation. 

Hypochlorhydria. 

This is a condition associated with excretion of the gastric juice showing 
0.1 or a lower per cent, of hydrochloric acid. It is found especially in subacute 
or chronic gastritis, in incipient carcinoma, in fevers, severe anemias, many 
mental diseases, passive congestion due to valvular heart lesions, many cases 
of chronic nephritis, and dilatation of the stomach. Some rare cases of ulcer 
of the stomach show a low degree of free hydrochloric acid, but this is not usual. 

Anachlorhydria. 

This is a condition characterized by the excretion of the gastric juice show- 
ing the complete absence of free hydrochloric acid. This condition has been 
supposed to be pathognomonic of gastric carcinoma. There are, however, many 
cases of cancer which show either a hypo- or a hyperchlorhydria, and there 
are, also, many other conditions which show an anachlorhydria. Thus we 



GASTRIC CONTENTS. 7 1 

find this condition in a large majority of cases of advanced chronic gastritis, 
in the severe anemias, especially of the pernicious type, in neurasthenia and 
hysteria, in many severe febrile diseases, and in atrophic gastritis. 1 

Hyperchlorhydria. 

This condition exists when we have the excretion of a gastric juice showing 
more than 0.2 per cent, hydrochloric acid. This figure may run anywhere 
from 0.2 to 0.9 per cent. 2 It is a very common occurrence in nervous individuals, 
in ulcer of the stomach, some cases of chlorosis, in some chronic cachexias, 
in the early stages of chronic gastritis, in carcinoma which is grafted on to an 
old ulcer, in continuous hypersecretion, in chronic passive congestion of the 
stomach, and in cases of migraine. 3 

It will thus be seen that the hydrochloric acid of the gastric juice varies 
under clinical conditions to quite an extent. While an increased amount of 
hydrochloric acid is usually present in ulcer of the stomach, we must not neces- 
sarily make our diagnosis on this point alone. Likewise in carcinoma of the 
stomach we should not exclude this condition if the examination of the stomach 
contents does not show a lessened amount of hydrochloric acid or even a total 
absence, as many cases of carcinoma may show all varieties of chlorhydria. 

(3). Combined Hydrochloric Acid. 

As the hydrochloric acid first secreted by the gastric glands combines with 
the protein of the food material, it is necessary to have some method by which 
we may determine just how much of this material has been formed in the 
stomach. The physiologically active hydrochloric acid consists of both the 
free and combined acid so that we may have only a slight amount of the free 
acid, but a relatively large amount of the combined. Not infrequently we find 
cases which show no free hydrochloric acid, but quite a percentage of the com- 
bined acid, indicating that a certain amount of acid has been secreted by the 
gastric juice. This combined hydrochloric acid has, therefore, a certain 
clinical i mportance and should be investigated in every case. 4 No direct methods 
are known for its determination so that we must resort to indirect methods. 

Method of Martius and Liittke. 

This method, as modified by Reissner, is much more applicable to scientific 
work than to clinical investigations, as it is too complicated and time-consuming 
for the ordinary practitioner. It is based upon the facts that the free hydro- 
chloric acid as well as the acid combined with protein material escape upon in- 
cineration of the gastric juice, while the inorganic chlorin in combination with 
inorganic bases remains in the ash. If the total amount of chlorin present 

1 Ohly (Deutsch. med. Wchnschr., 1913, XXXIX, 1402; Arch. f. Verdauungskr., 1915, 
XXI, 128) calls attention to the fact that 70 to 80 per cent, of all cases of progressive cholecystitis 
show an anacidity or actual achylia, although a slight hyperacidity may obtain in the incipient 
stages. This is of importance in differentiating a pyloric or duodenal ulcer from cholelithiasis. 
See, also, Hernando, Arch, des Mai. de l'App. digestif, 1914, VIII, 274; Griinfelder, Ztschr. f. 
exp. Path. u. Ther., 1914, XVI, 141. 

2 See page 69. 

3 Surmont and Dehon (Arch, des Mai. de l'App. digestif, 19 14, VIII, 246) believe that 
defective elimination of salt by the kidneys is often compensated for by hyperchlorhydria. 

4 See Grund, Deutsch. Arch. f. klin. Med., 1913, CIX, 560. 



72 DIAGNOSTIC METHODS. 

in the filtered gastric juice be determined (a), and then the amount of chlorin 
in the ash (b) investigated, subtraction of the latter (b) from the former portion 
(a) will give the amount of chlorin referable to free and combined hydrochloric 
acid except a small loss referable to volatilization of ammonium chlorid. If 
the gastric juice be neutralized with sodium hydrate before it is incinerated and 
the chlorin in this ash determined, the amount of this chlorin (a') subtracted 
from (a) represents the ammonium chlorid volatilized. Hence a'— b equals 
the free and combined HC1. By now determining the amount of free hydro- 
chloric acid according to Topfer's method we may at once calculate the combined 
hydrochloric acid by subtracting this result from the amount of free and combined 
hydrochloric acid previously obtained. The method of determining the chlorin 
will be fully discussed in the section on urine to which the reader is referred. 

Method of Topfer. 

This method embraces three separate determinations. In the first place the 
total acidity of the gastric juice is determined by titration of 10 c.c. of unfiltered 
gastric juice with tenth-normal sodium hydrate, using phenolphthalein as an 
indicator. This result is termed a. This indicator has the advantage of re- 
acting toward anything of an acid nature, and will give us, therefore, the dif- 
ferent factors which go to make up the total acidity of the gastric juice, namely, 
the free and combined acids, the organic acids, and the acid salts. 

Having determined this factor (a), a second portion of 10 c.c. of gastric 
juice is titrated with tenth-normal sodium hydrate solution using a i per cent, 
aqueous solution of alizarin (alizarin monosulphonate of sodium) as an indi- 
cator. Two or three drops of this indicator are added to 10 c.c. of unfiltered 
gastric juice when the mixture becomes distinctly yellow. The titration is 
carried to the point of production of a pure violet color which does not deepen 
on the further addition of alkali. As this reaction demands the recognition 
of a change from yellow through a faint violet to a deep violet color the worker 
must have considerable practice before he is able accurately to determine the 
end point. The result is termed b. No trace of red should be present, a pure 
violet color being the true reacting point. This color may be observed by 
treating a few drops of alizarin solution with a i per cent, solution of sodium 
carbonate. Alizarin reacts with free acid, both mineral and organic, and with 
acid salts, but not with organically bound HC1. If, therefore, we subtract the 
figure obtained when alizarin is used as an indicator (b) from that obtained with 
phenolphthalein (a) the result will be the combined hydrochloric acid (a— b). 

If now we add this combined hydrochloric acid to the free hydrochloric 
acid, which has been obtained by titration of the gastric juice using dimethyl- 
amido-azo-benzol as an indicator, (c), we obtain the total physiologically active 
hydrochloric acid (c+(a— b)). The difference between the total acidity and 
this factor gives us the amount of organic acid and acid salts present, (a — (c+ 
(a —b) ). If but a small amount of gastric juice be available for chemical ex- 
amination, recourse may be had to a modification suggested by Einhorn. This 
is a double titration of the same portion of juice. A few c.c. (5) of unfiltered 
gastric juice are treated with a few drops of dimethyl-amido-azobenzol and the 



GASTRIC CONTENTS. 73 

solution titrated for free HC1 with sodium hydrate. When the point of neutrali- 
zation of the free acid is reached a few drops of phenolphthalein solution are 
added and the titration continued to the point of neutralization of total acidity. 
These indicators do not interfere at all with one another as their reacting points 
are usually widely different. The writer has found this method reliable and 
convenient. 

Hydrochloric Acid Deficit. 

In those cases in which the gastric contents show no free hydrochloric acid, 
it is customary to determine the HC1 deficit. By this is meant the amount of 
hydrochloric acid which must be added to the gastric contents before it shows 
a reaction for free acid. This amount will naturally depend on the amount 
of combined HC1 already present, the amount of protein in the gastric contents 
and the amount of alkali secreted. Sahli suggests the expression "saturation 
deficit 11 for this figure. Ten c.c. of unfiltered gastric juice are titrated with 
tenth-normal hydrochloric acid, using dimethyl-amido-azobenzol as an indicator 
and titrating to the point of production of the red color. The result is expressed 
in terms of degrees as under the representation of the free hydrochloric acid. 
This factor enables one to follow the course of the disease, showing how little 
hydrochloric acid is excreted for combination with the proteins of the food. 

(4). Organic Acids. 

The organic acids, outside of the lactic acid, have very little clinical signifi- 
cance. The food practically always contains a certain amount of fatty acids 
which appear in the stomach contents and contribute to the total acidity. In 
the normal digestion of the carbohydrates, lactic acid is practically always 
formed, so that excess of this acid would indicate excessive fermentative proc- 
esses in the stomach, due to a combination of diminished amount of hydrochloric 
acid along with a lessened motility of the stomach. Other acids, such as buty- 
ric and acetic acids are formed in this same process of carbohydrate fermenta- 
tion, so that the organic acids may represent a large portion of the total acidity. 
Besides this, bacterial decomposition, in the absence of hydrochloric acid, plays 
a role in the production of these fatty acids. The fat-splitting ferment, lipase, 
may produce these organic acids in fairly large amounts. 

Total Organic Acid. 

It is sometimes of importance to know just how much organic acid is pres- 
ent in the stomach contents. This may be done directly by the Hehner-Maly 
method, which is based upon the fact that, if a mixture of organic and inor- 
ganic acids be neutralized and then incinerated, the organic acids will be con- 
verted into carbonates while the inorganic acids remain as neutral salts. If 
the alkalinity of these carbonates be then determined and this factor subtracted 
from the total acidity we obtain directly the mineral acids. This is possible 
owing to the fact that the degree of alkalinity of the carbonates is equal, in terms 
of tenth-normal solutions, to the acidity referable to the organic acids. The 
technic is as follows: The total acidity of 10 c.c. of gastric juice is deter- 
mined by titration with tenth-normal sodium hydrate solution as previously 



74 DIAGNOSTIC METHODS. 

described. The neutralized solution is evaporated to dryness in a platinum 
dish and is then incinerated. The ash is dissolved in distilled water and the 
alkaline solution titrated with tenth-normal oxalic acid solution. As i c.c. of 
the tenth-normal oxalic acid solution is equivalent to i c.c. of tenth-normal 
sodium hydrate solution, we subtract the factor obtained in the latter titration 
from that of the former and obtain directly the degree of acidity due to mineral 
acids. The number of c.c. of tenth-normal oxalic acid used represents directly 
the total organic acids present. 

In this method the acid salts are included in the factor referable to mineral 
acids, so that we may subtract from this factor the degree of acidity, attributable 
to free hydrochloric acid, and obtain the amount of acid salts present. In some 
cases fatty acids are present which are not soluble in water and consequently 
are not neutralized by the addition of the sodium hydrate solution. These 
acids may be extracted from the neutralized solution with ether and may then 
be neutralized and added to the neutral aqueous solution. The mixture is 
now evaporated as before and incinerated. This estimation of the higher fatty 
acids requires the use of the unfiltered gastric contents. However, such acids 
do not play a large clinical role and may be ordinarily omitted. 

(a). Lactic Acid. 

The ordinary foods such as milk, bread, and meat contain a certain 
amount of lactic acid, so that any test for the presence of lactic acid can be 
of value only when the meal contains very little of such foods or when the 
portion taken in with the food has disappeared from the stomach. After 
the Boas meal there is always less lactic acid than after the Riegel meal, 
so that the former is much preferable when a special test is to be made for the 
presence of lactic acid. Boas has shown that under physiological conditions 
no appreciable amount of lactic acid is formed during the process of digestion. 
At the height of digestion practically no lactic acid is demonstrable in the 
stomach contents. This may be due to its absorption, on the one hand, or, 
on the other, to the fact that the hydrochloric acid interferes with the delicacy 
of the reactions. Pathologically, lactic acid is found in any condition associ- 
ated with stagnation of the gastric contents as a result of motor insufficiency, 
provided the amount of hydrochloric acid is below the normal amount. As this 
condition of affairs is found most frequently in cases of carcinoma of the 
stomach, an excess of lactic acid is very strongly suggestive of malignancy, 
although it must be remembered that such an excess may appear in cases of 
benign stenosis and gastric insufficiency. If the stomach be washed out the 
evening before giving a test meal, preferably the Boas meal, and lactic acid 
be found in appreciable amounts, carcinoma is the probable diagnosis. This 
finding of increased lactic acid and diminished hydrochloric acid is not always 
observed in every case of carcinoma of the stomach. In some cases periods 
of increased production of hydrochloric acid alternate with increased formation 
of lactic acid, and in some cases, especially those in which the carcinoma has 
developed upon the base of an old ulcer, no lactic acid may be present, but 
hydrochloric acid may be found in large amounts. 



GASTRIC CONTENTS. 75 

Uffelmann's Test. 

This test is, perhaps, more commonly employed for the detection of lactic 
acid than is any other, but the writer prefers the Kelling test. Uffelmann's 
reagent consists of 20 c.c. of 1 per cent, carbolic acid solution, to which are 
added one drop of dilute ferric chlorid solution and sufficient water to form a 
transparent amethyst-blue solution. This solution is not permanent and must, 
therefore, be made fresh before each test. If a few drops of the filtered gastric 
juice be added to 5 c.c. of this reagent, the solution will be decolorized in the 
presence of lactic acid, taking on a beautiful canary-yellow or greenish-yellow 
tint. The mere decolorization of this solution is not sufficient for a positive 
test. A pure lemon yellow or canary color must be present before one may 
assume the presence of lactic acid. Even when this color appears one must 
eliminate such factors as the acid sodium phosphate, cane sugar, glucose, 
alcohol, and various organic acids, such as tartaric, citric, or oxalic, before he 
can say that lactic acid is present. A considerable excess of hydrochloric acid 
in the gastric juice may prevent the appearance of this color and likewise a 
yellowish tint of the stomach contents may obscure the result. Under such 
conditions it is necessary to extract the gastric contents with ether, which 
takes up the lactic acid, leaving the interfering substances behind. The 
ethereal solution is then evaporated, the residue taken up with distilled water, 
and the test applied to this solution. 

Kelling's Test. 

This test is in reality a modification of the previous one and consists in the 
addition of a few drops of filtered gastric juice to a very dilute solution of ferric 
chlorid. As used in the writer's laboratory, the method is as follows: To 
a test-tube full of water are added one or at most two drops of a 10 per cent, 
solution of ferric chlorid. The mixture is thoroughly shaken and divided into 
two portions, one of which serves as a control. On now adding a few drops 
of filtered gastric juice to one of these portions a distinct canary-yellow color 
will appear in the presence of lactic acid. The color of the two solutions 
should be compared so that any change in the one, to which gastric juice was 
added, may be observed. This test has the same objections as the Uffel- 
mann test, so that it is always wise to extract the gastric juice with ether before 
applying either one of these tests. 

Strauss' Method. 

This method is, perhaps, the very best clinical method at our disposal, 
as it shows lactic acid when present in pathological amounts. It does not, 
however, give a quantitative result, nor does one seem necessary in the ordinary 
clinical work. Into a special separatory funnel (see cut) are introduced 5 c.c. 
of the gastric juice. The funnel is then filled to the 25 c.c. mark with alcohol- 
free ether and well shaken. The ethereal layer will take up the lactic acid 
from the gastric contents. After the fluids have settled the gastric juice and 
ether are allowed to run out to the mark 5 by opening the stop-cock, after 
which distilled water is added to make up the 25 c.c. volume. Two drops 
of 10 per cent, ferric chlorid solution are then added with a medicine dropper 



76 



DIAGNOSTIC METHODS. 



and the mixture well shaken. The water will now extract the lactic acid 
from the ether. The aqueous layer is colored an intense greenish -yellow 
if more than o.i per cent, of lactic acid is present, while smaller amounts 
will show a slight greenish tinge. This test may be negative if the lactic 
acid present is completely combined with the proteins of the gastric juice. In 
such cases hydrochloric acid may be added to liberate this lactic acid before 
shaking out with ether. 

Other qualitative tests as well as several quantitative 
tests have been given for lactic acid. Quantitative deter- 
minations of lactic acid do not seem to be of any great 
clinical importance, as any marked reaction for this sub- 
stance is indicative of a pathologic condition whose extent 
may bear no relation whatever to the amount of lactic acid 
present. A general idea of the amount of lactic acid may 
be obtained by evaporating 10 c.c. of gastric juice, acidulated 
with a few drops of sulphuric acid, to the consistency of a 
syrup and then extracting this residue several times with 
acid-free and alcohol-free ether. The ether may be re- 
moved by evaporation and the residue taken up with 
water. This watery solution may now be titrated with 
tenth-normal sodium hydrate, each c.c. of alkali used repre- 
senting 0.009 gram of lactic acid. 

The method of Boas, while very exact, is much too 
complicated and time-consuming for clinical work so that 
the writer will refer to other books for a description of 
this test. The principle of the method is based upon the 
fact that when lactic acid is heated with a strong oxidizing 
agent it is decomposed into acetic aldehyd and formic acid. 
If now the aldehyd be distilled off and transformed into 
iodoform by the addition of alkaline iodin solution, this 
iodoform may be quantitatively determined. 



Fig. 17. — Strauss' 
s eparatory funnel. 
(Hemmeter.) 



(b). Butyric Acid. 

This acid does not occur in the gastric contents, under physiological con- 
ditions, unless much milk or carbohydrate food has been introduced. Fliigge 
has shown that butyric acid may be derived from lactic acid and conse- 
quently may be present under the same conditions in which lactic acid is 
found. As butyric acid may be introduced from without and may have been 
formed in the mouth, one should be careful in drawing conclusions as to the 
clinical significance of butyric acid. 

If present in any large amount, butyric acid may be usually recognized by 
its distinct odor which is that of rancid butter. This test may not be sufficient 
for the recognition of butyric acid so that it is advisable to shake out the gas- 
tric juice with ether, evaporate, and take up with water as described under Lactic 
Acid. If a small pinch of powdered calcium chlorid be added to this watery 
solution and the mixture warmed, butyric acid will separate from the fluid in 



GASTRIC CONTENTS. 77 

the form of small fat drops which float on the surface and have a characteris- 
tic odor of rancid butter. 

If a portion of the dried ethereal extract of the gastric juice be treated with 
a few drops of concentrated sulphuric acid and a little alcohol, the odor of ethyl- 
butyrate is perceptible on slight warming. This odor is the peculiar one of 
pineapples and is very easily recognized. This test is known as the pine- 
apple test. 

(c). Acetic Acid. 

It is not an infrequent occurrence to find acetic acid in rather large amounts 
in pathological conditions. The pathologic acetic acid is formed by the 
bacterial decomposition of the alcohol which is produced by the action of yeast 
upon carbohydrates. As yeast fungi are so frequently present in cases of dilata- 
tion of the stomach, associated with stagnation of its contents, acetic acid may be 
found under such conditions and may constitute a large portion of the total 
acidity. 

In testing for acetic acid the aqueous extract of the ethereal residue of 
the gastric juice is carefully neutralized with sodium carbonate solution. 
If a few drops of ferric chlorid solution be added to this neutralized solution 
a deep red color will appear if acetic acid be present. If this solution be 
boiled a reddish precipitate of basic ferric acetate is formed. This neutrali- 
zation of the aqueous solution is an essential point in this test, as the presence 
of free acid will prevent the appearance of any precipitate and the presence 
of free alkali will cause the formation of ferric hydroxid which will mislead, 
as the coloration is very much the same. The writer has seen several cases in 
which acetic acid was mistaken for lactic acid when the Kelling test was applied. 
On adding gastric juice containing a large amount of acetic acid to the dilute 
ferric chlorid solution a change in color is observed, but in no case do you get 
the distinct canary-yellow color which can be possibly referable to acetic acid. 

(5). Gastric Ferments. 
(a). Pepsin. 

The enzyme pepsin is the most important of the ferments occurring in 
the gastric juice. As previously stated this ferment is excreted in the form of the 
zymogen (pepsinogen or propepsin) by the chief cells of the fundus glands. 
It becomes active, that is, converted into pepsin, by the free hydrochloric acid 
of the gastric juice. This ferment acts only in acid media and is destroyed 
by the presence of minute traces of alkali. 1 Its action is continuous, a small 
portion being capable of digesting large amounts of albumin, providing the 
products of this digestion are gradually removed. Should the products of 
ferment activity remain in the stomach an undue length of time, this fer- 
ment will cease to be active owing to the accumulation of the products of its 
own activity. Pepsin acts in the presence of many other acids, but the concen- 
tration of these acids must be higher than in the case of hydrochloric acid. 
Thus, a 0.2 to a 0.4 per cent, hydrochloric acid gives the best results with 

1 Hamburger (Arch. Int. Med., 1915, XVI, 356) confirms the fact that sodium chlorid 
inhibits the action of pepsin as formerly suggested by Schiitz and Levites. See, also, Edie 
(Biochem. Jour., 1914, VIII, 193) for a discussion of the reciprocal action of pepsin and trypsin. 



78 DIAGNOSTIC METHODS. 

pepsin, while a 1 to a 1.018 per cent, lactic acid is necessary to bring about 
good results. 

Very little data exists as to the amount of pepsin or of its zymogen so that 
we are forced to draw our conclusion regarding a normal or abnormal amount 
of this ferment from the rate at which known amounts of albuminous material 
are digested. Pepsin acts only upon protein substances, giving rise to a 
series of decomposition products which will be discussed later. Normally 25 
c.c. of gastric juice will dissolve (digest) 0.05 to 0.06 gram of serum albumin 
in one hour, the same amount of coagulated egg-albumin in three hours, and 
a similar amount of fibrin in one and one-half hours. 

A diminution in the amount of pepsin must be referable to a direct disease 
of the secreting gland, as general abnormalities do not affect this function 
as much as they do the production of free hydrochloric acid. 1 Pepsin is usually 
present when the free hydrochloric acid is either increased or diminished, but 
in cases of carcinoma, atrophic gastritis, and in occasional cases of pernicious 
anemia we may find no pepsin and no hydrochloric acid. Such a condition is 
known as achylia gastrica and occurs sometimes as a direct pathologic con- 
dition without a known etiology. It frequently happens that pepsin is present 
when no hydrochloric acid is found. 

Qualitative Tests. 

The digestive power of the filtered gastric contents will depend, of course, 
upon the amount of pepsin and the amount of free acid present. Artificial 
digestion experiments are at present the only methods by which we may test 
the amount of pepsin. The substances used in these digestion experiments 
are egg-albumin and fibrin. The fibrin may be prepared by beating freshly 
drawn ox-blood with a glass rod until the coagula are distinctly formed. These 
stringy masses are washed thoroughly in water to remove the coloring matter 
of the blood, are then cut into small pieces of uniform size and are kept in al- 
cohol for a few days. The. c e hardened masses are placed for one to two days 
in a neutral concentrated solution of carmin. They are then washed in water, 
thoroughly pressed, and are preserved in glycerin to which a little carmin has 
been added. Before being used they should be washed in water to remove the 
glycerin and free coloring matter. The egg- albumin is prepared for these 
experiments by boiling an egg until the albumin is distinctly coagulated. This 
material is then cut into cylinders of about 5 mm. in diameter with a cork borer 
and are then sectioned into disks 1 mm. thick. These disks may be preserved 
in glycerin. 

In testing for the presence of pepsin 25 c.c. of gastric juice, which must 
contain free hydrochloric acid, are placed in a flask which contains a few pieces 
of fibrin or a disk of coagulated egg albumin. This flask is then placed in the 
incubator from 37 to 40 and allowed to remain until the protein is completely 
dissolved. If pepsin is present the fibrin will show signs of digestion by swelling 
up in from 15 to 30 minutes, the egg-albumin in from one-half to one hour. 
Within an hour and a half the fibrin should be practically dissolved, while the 
egg-albumin will require about three hours. 

1 See Carlson, Am. Jour. Physiol., 1915, XXXVIII, 248. 



GASTRIC CONTENTS. 79 

If no hydrochloric acid is present in the gastric juice a few drops of 10 per 
cent, hydrochloric acid are added to 25 c.c. of gastric juice and the test per- 
formed in the same manner. A positive result will indicate the presence of 
the zymogen, pepsinogen. 

Quantitative Examination. 

Certain laws have been discovered regarding the action of ferments in 
general and these are applicable to pepsin. Schiitz has found that the relative 
quantities of pepsin in digesting mixtures containing the same quantity of 
hydrochloric acid are proportional to the squares of the quantities of albumin 
digested in the same time, or, in other words, the activity of a ferment varies as 
the square root of its amount. Nirenstein and Schiff 1 have found that this law 
applies only for the less concentrated pepsin solutions. If the quantity of 
pepsin in the digesting fluid is so large that more than 3.6 mm. of albumin 
(see Mett's Test) are digested in 24 hours, the above law does not give reli- 
able values for the quantity of pepsin. The gastric juice under such circum- 
stances must be diluted before this law applies. Sahli rightly calls attention 
to an important reason for diluting the gastric juice before testing for pepsin, 
namely, the constant presence of the products of peptic digestion which inhibit 
further peptic activity. The gastric juices which contain a diminished amount 
of hydrochloric acid are the richest in these inhibiting substances and should 
be carefully studied. The presence of these substances gives rise to conditions 
which make it impossible to arrive at accurate conclusions if the pepsin value 
is calculated from the pure gastric juice. 

Hammerschlag's Method. 

Ten c.c. of a 1 per cent, filtered solution of egg-albumin in 0.4 per cent. 
HC1 are poured into two test-tubes. As fresh egg-albumin contains about 13 
per cent, of dry protein, it should be diluted about 13 times to make a 1 per 
cent, solution. To one of the test-tubes 5 c.c. of gastric juice are added, to 
the other 5 c.c. of distilled water, both being placed in the incubator at body 
temperature for one hour. At the end of this time the albumin in each tube is 
estimated by Esbach's method (see Urine). The difference between the pre- 
cipitate of albumin in the two tubes is equal to the amount of albumin which 
has been digested and forms, therefore, a measure of the peptic activity of the 
gastric juice, the square root of the amount of pepsin being proportionate to 
the quantity of albumin dissolved. This test is open to the objection that the 
albumin of the gastric juice, as well as the albumoses are precipitated by the 
reagent. In one hour not all of the egg-albumin will be digested, normally 
only about 90 per cent. 

Mett's Method. 

This method is, perhaps, the one most frequently used and the one which 
gives quite as accurate results as the others advised. 2 The whites of several 
eggs are mixed, in order to avoid accidental variations in the egg-albumin, 

1 Arch. f. Verdauungskr., 1903, VIII, 559. 

2 See Christiansen, Biochem. Ztschr., 1912, XLVI, 257; also, Waldschmidt, Arch.f. d. ges. 
Physiol., 1912, CXLIII, 189. 



80 DIAGNOSTIC METHODS. 

and are filtered. The gas should be removed from this material by the use of 
a suction pump as far as possible. A number of glass capillary tubes, each from 
10 to 30 cm. in length and 1 to 2 mm. in diameter, are then filled by suction 
with]this albumin. They are then laid in the bottom of a vessel which is placed 
for five minutes in boiling water in order to coagulate the albumin. The tubes 
are taken out, wiped carefully, and the ends sealed with paraffin or sealing 
wax. It occasionally happens that these tubes contain air-bubbles, which will, 
however, disappear in a few days. These longer tubes may be kept in stock 
for a considerable period of time. In performing the test for pepsin by the use 
of these tubes of coagulated albumin, the longer tubes are cut into lengths of 
about 2 cm. and are placed in a small dish or watch-glass with 5 c.c. of the 
gastric juice, which must necessarily be acid in reaction. These dishes con- 
taining the gastric juice and filled glass tubes are then placed in the incubator 
for 10 to 24 hours. At the end of this time the length of the digested column 
at each end of each tube is measured and the average length of the column 
of albumin digested estimated. The square of this digestion length is the 
measure of the relative amount of pepsin in the gastric juice. The unit upon 
which one may base comparative results of the relative amount of pepsin is 
that quantity of pepsin by which 1 mm. of albumin in a Mett's tube will 
be digested in 24 hours with an acidity of 0.18 per cent, free HC1. The length 
which theoretically pure pepsin would give is 4 mm. the units represented by 
the pepsin of the gastric juice being anywhere from o to 256. 

Nirenstein and Schiff, for the reasons previously mentioned, advise 
the dilution of a gastric juice before applying this test, believing that a 
dilution of 16 will give more nearly exact quantitative results. Their method 
is as follows: One c.c. of the filtered gastric juice is diluted with 16 c.c. of 
0.18 per cent. HC1. The procedure is then the same as in Mett's method, 
the results being multiplied, of course, by 16 in order to obtain the actual num- 
ber of units in the gastric juice. In some cases it has been found that a dilution 
of 1 to 32 gives better results, but this is rather unusual. The results of these 
workers show that striking differences exist in individual gastric secretions, 
figures ranging between o and 256 units being obtained. This points to the 
fact that the pepsin concentration is independent of the amount of acid in the 
gastric juice. The quantitative estimation of pepsinogen may be carried out 
by either one of the methods previously outlined for pepsin by rendering the 
gastric juice acid with hydrochloric acid up to 1 to 2 parts per thousand. 

Method of Thomas and Weber. 

This method 1 is based upon the digestion of an acid casein solution in 0.2 
per cent, hydrochloric acid by the gastric juice. The acid casein solution is 
prepared by dissolving 100 grams of finely powdered dry casein in 1,900 grams 
of a solution of hydrochloric acid containing 5.04 grams HC1. Five c.c. of 
gastric juice are added to 100 grams of the casein solution. Dilute with dis- 
tilled water to 250 c.c. and place in the incubator. After the end of one hour 
the digestion mixture is poured into 100 c.c. of 20 per cent, sodium sulphate 

1 Centralbl. f. Stoffwechsel. u. Verdauungskr., 1901, II, 365. 



GASTRIC CONTENTS. 8 1 

solution, in which the non-digested casein is completely precipitated. This 
is collected on a weighed filter and washed with distilled water until no trace 
of sulphate reaction is evident. It is then dried with alcohol and ether and 
weighed. The difference in weight between this undigested residue and that 
contained in the original amount taken gives the amount of casein digested. 

This method, as modified by Volhard, consists in the titration of the acid- 
ity of the filtrate from the solution to which the sodium sulphate has been 
added. The total acidity is higher, the more the casein is in the uncoagulated 
form and the increase in the acidity will vary as the square root of the amount 
of pepsin. 1 

(&). Chymosin (Rennin). 
The normal gastric juice contains a second ferment, chymosin, which 
has the function of coagulating milk independently of the presence of acid. 
The zymogen of this ferment becomes active, however, only in the presence of 
acids; that is, the zymogen is converted into rennin by acids. In this process 
of coagulating milk, insoluble casein is formed from the caseinogen of the milk 
by the combined action of the rennin ferment and calcium salts, while the 
curdling of milk is due to the precipitation of unchanged caseinogen by acids. 
It is evident, therefore, that this process resembles very closely that of the co- 
agulation of the blood, as calcium salts are absolutely essential for its success. 
Whether rennin is identical with pepsin, as some believe, is still an unsettled 
question. 2 

Leo's Method. 

Three to five drops of gastric juice are added to 5 or 10 c.c. of fresh 
uncooked neutral or amphoteric milk and the mixture placed in the incubator 
for 15 to 20 minutes. If rennin is present in normal amounts coagulation will 
be observed. In this process one may not be sure as to whether the curdling 
is due to the action of rennin or to that of the acid. Rennin action occurs 
typically only when no change in the reaction of the milk has taken place. 

RiegePs Method. 

Three to 5 c.c. of neutralized gastric juice are added to 5 to 10 c.c. of 
fresh milk. This mixture is placed in the incubator and left for 15 minutes, 
when distinct coagulation will occur in the presence of rennin. If the milk 
be boiled previous to treatment the result is not so typical. 

Quantitative methods for the determination of rennin are at present 
uncertain and are even of doubtful utility. So little is known about the varia- 
tions in the rennin ferment of the gastric juice that an exact determination would 
add little to the clinical history. According to Glassner, pepsin and rennin are 
both diminished in cases of tumor of the fundus, while pepsin is diminished and 
rennin normal in tumors of the pylorus. 

1 For other methods see Jacoby and Solms, Ztschr. f. klin. Med., 1907, LXIV, 159; Fuld 
and Levison, Biochem, Ztschr., 1907, VI, 473; Hata, Ibid., 1909, XXIII, 179; Neilson and Bon- 
not, Arch. Int. Med., 1913, XI, 395; Geselschap, Ztschr. f. physiol. Chem., 1915, XCIV, 205; 
Spencer, Jour. Biol. Chem., 19 15, XXI, 165. 

2 See Fuld, Internat. Beitr. z. Path. u. Therap. d. Ernahrungsstor., 1913, V, 104. 

6 



82 DIAGNOSTIC METHODS. 

(c). Lipase. 

While it is undoubted that lipase occurs in the gastric secretion, its action 
is very slight. Normally, gastric digestion is not much concerned with the 
splitting of fat into lower products, but, as Volhard has shown, this action does 
occur. In testing for the presence of lipase in gastric contents it is necessary 
that the examination be made after the stomach is thoroughly washed out 
following the administration of a test meal free from fat. 

This ferment may be detected qualitatively by adding a small piece of 
fresh neutral butter to the gastric juice and placing the mixture in the incubator 
for one hour. At the end of this time a distinct odor of butyric acid will be 
observed. 

It may be said that owing to this lipolytic action statements have arisen 
that the Sahli test meal gives erroneous results. Careful work by Seiler shows 
that the amount of fat decomposed under the conditions of the test meal is so 
slight that it may be neglected. Volhard found that after two hours from 30 
to 36 per cent, of the fat was split up into fatty acids, which aid in dissolving 
the bile and in forming an emulsion with the neutral fat in the intestinal canal. 

The Products of Protein Digestion. 

It is generally stated that the action of the pepsin and hydrochloric acid 
of the gastric juice upon protein material passes through the following stages: 
The albumin is first converted into acid-albumin (syntonin), then into albu- 
moses,of which there are four (prot-albumose, hetero-albumose, dys-albumose, 
and deutero-albumose), and ultimately into peptone. It is well established 
that pepsin itself will not carry the hydrolysis beyond the stage of peptone or 
somewhat lower polypeptids. The finding of amino-acids and hexon bases, 
by the author as well as by others, in the contents of a dilated stomach must 
be interpreted as meaning a conversion of the pepsin products into lower 
cleavage units through the action of some other ferment, whether derived 
from the intestinal canal or excreted into the stomach from a malignant growth. 
Pepsin has, therefore, a function distinct from that of trypsin, attacking different 
linkings within the protein molecule. 

It is practically never necessary to test the stomach contents for such 
material in clinical work, so that the writer will refer to text-books on physio- 
logic chemistry for a description of the products of gastric protein digestion. 

The Products of Carbohydrate Digestion. 

The pure gastric juice, owing to its acid content, inverts sugars to a certain 
extent. The amount of this inversion depends on the number of free hydro- 
gen ions arising from the acid, and is only very slight under normal conditions. 

As previously described, the saliva converts starch into soluble substances 
through the stage of soluble starch, erythrodextrin, achroodextrin and finally 
maltose. The action of the ptyalin of the saliva is inhibited by the free acid 
of the gastric juice, but the action of this salivary ferment is so rapid that from 
50 to 75 per cent, of the starch is converted into a soluble form. While these 
products are not ordinarily tested for in the gastric contents, the fact of their for- 
mation and presence must be remembered. An excess of starchy material 



GASTRIC CONTENTS. 83 

in the food will lead to an increased amount of such decomposition products 
in the stomach when the acid of the gastric juice is not present in sufficient 
amount to inhibit the action of the ptyalin of the saliva. 

Blood. 

Blood is not a normal constituent of the gastric contents, but is found fre- 
quently in conditions associated with erosion, in ulcer, and in carcinoma. 
The appearance of the blood in cases of ulcer is usually that of fresh bright 
red blood, which may, however, be changed to a brownish substance due to the 
action of the excess of acid commonly present in this condition. In carcinoma 
the blood is more intimately mixed with the stomach contents and appears in the 
form of brownish-black clumps, constituting the so-called "coffee-ground" 
material. The tests for the presence of blood will be given in detail under 
Feces, to which the reader is referred. 

Gases. 

The stomach usually contains a certain amount of gas which may have 
been swallowed, may have passed into the stomach from the duodenum, or 
may have been produced in the stomach by processes of fermentation. The 
examination for these gases is not of great clinical importance, but a general 
idea of the different kinds of gases present seems essential. During the proc- 
esses of normal digestion, nitrogen, oxygen, and carbon dioxid may occur from 
the protein digestion, while hydrogen, marsh-gas, and olefiant gas may arise 
from the carbohydrate hydrolysis. In abnormal processes of digestion 
we may find ammonia and hydrogen sulphid arising from decomposing protein 
material. The work of Boas seems to indicate that the hydrogen sulphid is 
more commonly present in cases of benign gastric dilatation and is rare in car- 
cinoma. This hydrogen sulphid does not seem to be produced either in the 
presence of free hydrochloric or of lactic acid. The presence of hydrogen sul- 
phid can, however, not be considered as a specific substance in the stomach 
contents, as Dauber has shown that almost every stomach contains bacteria 
which may produce this gas from sulphur-containing bodies. In cases of dila- 
tation of the stomach, providing the motility be sufficiently diminished, we 
find fermentation with resulting gas production even though hydrochloric 
acid be present. Such a condition never occurs if the motility is normal, utterly 
regardless of the amount of hydrochloric acid, as in cases of diminished hydro- 
chloric acid lactic acid will usually prevent such a process. 

One may show the presence of the gases in the stomach contents by filling 
a fermentation tube with the well-mixed unfiltered gastric contents and placing 
it in the incubator for some time. If there is no gas within 24 hours it may be 
wise to wait at least 48 hours to permit of the proper diffusion of the gas. If 
gas is formed its nature may be determined by the ordinary chemical tests. 
This test has some value in determining the degree of stagnation of the stomach 
contents, but it must be remembered that a small amount of gas is contained in 
the normal stomach. 

Other substances, such as acetone, have been found in the gastric contents 
in pathologic conditions, but tests for these substances are rarely of importance. 



84 DIAGNOSTIC METHODS. 

In conditions associated with the presence of acetone in the stomach contents 
this substance is usually detected in the breath. 

Functions of the Stomach and its Contents. 

The stomach is to be regarded as a specialized portion of the alimentary 
tube in which the first stages of digestion of protein material take place. This 
occurs under the combined action of the hydrochloric acid and pepsin, the 
resulting products being gradually passed into the duodenum through the py- 
lorus. An increased acidity of the gastric juice may be associated with a distinct 
spasm of the pylorus so that food material cannot pass into the duodenum. 
On the other hand, a lessened degree of acidity is associated with hypermotility, 
the contents passing rapidly into the intestine, where it is acted upon by the pan- 
creatic ferments. Beside the function of digestion, principally of the protein 
foods, the stomach serves as a reservoir to hold the food material, allowing it to 
pass only in small portions into the bowel at any one time. 1 Owing to the pres- 
ence of hydrochloric acid, the gastric juice is antiseptic, rendering inert many 
but not all types of bacteria. Moreover, the hydrochloric acid activates the 
zymogens and thus permits of action upon all types of food material. The 
work of Pawlow has shown that the acid of the gastric juice is one of the most 
powerful stimulators of pancreatic secretion. The mechanism of correlation 
between the stomach and bowel is more easily understood if this point be 
borne in mind. The acid chyme coming from the stomach is poured out only 
in small portions at a time so that the pancreatic juice secreted may act upon 
the smaller portions as they are passed into the bowel. 

VI. Motility of the Stomach. 

It is probably true that disturbances in the motility of the stomach are in 
reality of more importance than are those in the secretory activities. 2 Under 
normal conditions of motility the food material passes into the intestine and is 
digested there, although no previous gastric digestion has taken place. If 
the motility be much impaired, stagnation of food with resulting dilatation of 
the stomach will occur, which will give rise to more or less serious disturbance. 
The motor disturbances are of three types, (i) vomiting, (2) hypermotility, 
and (3) motor insufficiency. The most important of these is the latter, as 
the former has little influence upon actual digestion in the stomach, although 
the patient may suffer for want of adequate nutrition; while in hypermotility 
the gastric disturbance will not be much noticed, owing to the fact that the food 
is rushed into the duodenum where it is digested. The consequences of motor 
insufficiency may be either disorders of secretion, decomposition, or both. 
Simple pathologic conditions which hinder the emptying of the stomach, such 
as ulcers, cicatrices, spasm of the pylorus, and simple atony, are usually asso- 
ciated with hyperacidity, while malignant conditions usually show a dimin- 

1 Tangl and Erdelyi (Biochem. Ztschr., 1911, XXXIV, 94) and Fejer (Ibid., 1913, LILT, 
168) have shown that the fats are discharged into the bowel at different rates depending upon 
their melting point and their viscosity. The higher the melting point the longer a fat remains 
in the stomach, while the more viscid a fat the less does it retard the discharge of non-fat foods. 

2 See Carlson, Am. Jour. Physiol., 1912, XXXI, 151. 



GASTRIC CONTENTS. 85 

ished secretion. 3 It is quite rare to find a case of motor insufficiency without 
disorder of secretion, rarer at all events than disorders of secretion without motor 
disturbances (Schmidt). Motor insufficiency is quite commonly followed by 
decomposition of the gastric contents and may even be considered the chief 
cause of such decomposition. Under these conditions of disturbed secre- 
tory and digestive activity associated with motor insufficiency we find the absorp- 
tive power of the stomach very much affected. 

Hypermotility is seen in many cases of hyperacidity, but it must be 
remembered that primary hyperacidity may cause spasm of the pylorus and 
hence bring on a distinct motor insufficiency. We cannot, therefore, judge of 
the motility of the stomach from the degree of acidity of the gastric 
contents. An enlarged stomach is not necessarily associated with motor in- 
sufficiency. Cases of megalogastria are more or less frequent in which the 
motility is practically normal. When a dilatation is associated with motor 
insufficiency it is clinically styled an ectasia or ectasis, being known as atonic 
gastric ectasis if the condition is due to weakness of the muscle, while it is styled 
hypertonic gastric ectasis if due to pyloric stenosis. Normally, no food should 
be found in the stomach within seven to eight hours after taking, no matter 
how large the meal. 2 We have, therefore, a method by which we may judge 
of the motive power of the stomach. 

Leube's Method. 

Leube administers a Riegel test meal and washes out the stomach with a 
liter of water six hours after. If only very slight traces of food are found in the 
washings .the motor power is regarded as normal. 

Boas' Method. 

Boas administers a simple evening meal consisting of meat, bread and 
butter, and tea, washing out the stomach the following morning. If any food 
material is found the motor insufficiency is considerable. If the stomach be 
washed out previous to the administration of the evening meal no food should 
be found in the stomach in the morning. 

Method of Ewald and Sievers. 

This test is based upon the observation that salol is decomposed into car- 
bolic and salicylic acids only in an alkaline medium. As the salicylic acid is 
eliminated in the urine in the form of salicyluric acid, it is possible to determine 
the rate of passage of salol from the stomach to the small intestine. It seems 
necessary to state that the assumptions on which this test are based are partially 
wrong. In the first place salol is split into its constituents by gastric juice 
within 15 minutes, although the degree of dissociation is slight. Moreover, a 
certain amount of absorption of salicylic acid takes place from the stomach 
so that a reaction may be obtained in the urine within 15 minutes in cases 
in which no hypermotility exists. 

One gram of salol is given to the patient immediately after a meal. The 
urine is then collected every 15 minutes for two hours and tested by the addi- 

1 See Hamburger and Friedman, Arch. Int. Med., 19 14, XIV, 722. 

2 See Faulhaber, Berl. klin. Wchnschr., 1914, LI, 1355. 



86 DIAGNOSTIC METHODS. 

tion of a small amount of ferric chlorid solution, which will give a violet 
color in the presence of salicyluric acid. 

Under normal conditions, according to Ewald, a positive reaction occurs 
in from 45 to 75 minutes. A further delay above 75 minutes is indicative of 
motor insufficiency, the degree of insufficiency bearing some relation to the 
time of appearance of this reaction. Should no result be obtained after 24 
hours a stenosis of the pylorus is highly probable. 

As the writer has so frequently found a reaction for salicyluric acid in the 
urine within 15 minutes, which is due not to the action of the hydrochloric 
acid or the ferments of the gastric juice, but to the moisture, temperature, and 
bacteria, he is accustomed to use the time at which a reaction for this substance 
disappears from the urine rather than the time at which it makes its first ap- 
pearance, as the basis of judgment regarding the motility of the stomach. 
Normally, no reaction for salicyluric acid should occur in the urine after 24 
hours although Huber states that it may take 26 to 27 and hence limits his 
time to these latter figures. It might be wise to follow the suggestion of Sahli 
and determine both the time of appearance and disappearance. 

Sahli has called attention to the fact that we are not justified in assuming 
a pyloric stenosis in case food material is found in the stomach even several 
days after being taken. He adds that the emptying of the stomach is regu- 
lated by the intestine rather than by the stomach itself, since nutritive substances 
reaching the intestine effect a reflex closure of the pylorus (von Mering's 
reflex) until the intestine has completed its work. The motor activity of the 
stomach should, therefore, be examined under conditions in which this reflex 
does not occur. This he determines by estimating the length of time re- 
quired by the stomach to empty itself of a half-liter of water, the stomach being 
thoroughly washed out previously. 1 

Winternitz Test. 

Winternitz has recommended the use of iodipin instead of salol for testing 
the motility of the stomach. This substance is not affected by the gastric 
contents, but is acted upon in the intestine by the pancreatic secretion and bile 
in such a way that iodin is set free. This may be tested for in the saliva by 
adding to it a little starch paste, when a distinct blue color will be observed 
within 15 to 45 minutes. 

VII. Absorptive Power of the Stomach. 

The absorptive power of the stomach is not of great importance clinically, 
as the greatest part of absorption occurs from the intestinal tract. However, 
tests for such power have a certain associated value and are, therefore, usually 
made. For this purpose Penzoldt and Faber advance a method depending 
upon the principle that under physiologic conditions potassium iodid is rapidly 
absorbed by the gastric mucous membrane and is immediately eliminated in 
the saliva. A capsule containing two to three grains of potassium iodid is 

1 See Boas (Deutsch. med. Wchnschr., 1912, XXXVIII, 455), Kemmerling (Arch. f. 
Verdauungskr., 1914, XX, 49) and Wartensleben (Ibid., 66) for a discussion of the Boas, 
chlorophyl test. 



GASTRIC CONTENTS. 87 

given to a patient shortly before a meal. The saliva is then tested as follows 
for the presence of potassium iodid at intervals of two to three minutes. The 
saliva is slightly acidified With nitric acid and treated with a few drops of starch 
paste when the characteristic blue color of iodid of starch will be formed by 
the action of the iodin liberated from the potassium iodid by the nitric acid. 
Under physiological conditions the first trace of iodin will appear in the 
saliva within ten minutes of its administration upon an empty stomach. Under 
pathological conditions a delayed reaction may be observed in almost all dis- 
eases of the stomach, especially in dilatation and in carcinoma. The test will 
naturally be delayed in case the stomach is filled with food. This test has little 
value, as it may appear or not in all types of gastric disease. Von Mering has 
found that potassium iodid is not absorbed at all from the stomach even within 
two or three hours, so that the iodin appearing in the saliva may be due to 
absorption from the intestine. 1 

VIII. Indirect Examination of the Stomach Contents. 

As not all cases of disease of the stomach permit of examination by removal 
of the contents through the stomach-tube, methods have been advanced to 
permit of indirect determination as to the activity of the stomach contents. 
These methods do not permit of accurate determination of the acidity or of the 
ferments of the juice, but do give much information regarding the normal 
digestive powers and motility of the stomach. 

Giinzburg's Method. 

A tablet of 0.2 gram of potassium iodid is placed in a piece of the thinnest 
possible strongly vulcanized rubber tubing measuring about 2.5 cm. in length. 
The ends of the tubing are folded and the package tied with three threads of 
fibrin which have been hardened in alcohol. The package is now tested by 
placing it in warm water for several hours and examining the water for potas- 
sium iodid. The patient swallows one of these packages three-quarters of 
an hour after an Ewald meal, the saliva being tested for potassium iodid at 
intervals of 15 minutes. In the presence of free hydrochloric acid in normal 
amounts the threads of fibrin are dissolved and the potassium iodid is absorbed, 
giving a reaction in the saliva in from one to one and three-quarters hours. In 
cases of hypochlorhydria the reaction is delayed, a delay of six hours indicating 
a practical absence of free hydrochloric acid. 

This test very frequently gives reliable results, but the threads of fibrin 
soon become brittle and break on swallowing the package so that a reaction for 
potassium iodid under these conditions would have no value. 

Sahli's Desmoid Reaction. 

Sahli has recently introduced the "Desmoid bag" for use in estimating 
the functional activity of the stomach. These bags are made of the ordi- 
nary rubber-dam used by dentists and contain a pill of 0.05 gram of methy- 
lene blue and 0.1 gram of iodoform. The bag is tied, in a manner especially 

1 See Mendel and Baumann (Jour. Biol. Chem., 1915, XXII, 165), who show that absorption 
is not a prominent function of the stomach. 




88 DIAGNOSTIC METHODS. 

outlined by Sahli, with cat-gut which has been allowed to dry but has been 
untreated chemically. This gut, according to Sahli, is digested only by the 
gastric juice and not by the pancreatic juices. This pill is administered to 
the patient immediately following the noon meal and the urine and saliva 
tested at intervals of one hour, beginning three hours after administration of 
the pill. The digestion of the gut by the gastric juice liberates the pill and 
permits of the absorption of both the methylene blue and the iodoform. The 
methylene blue will appear in the urine coloring it green within six hours, 
while the iodin will be found in the saliva within two hours. Should the 
color of the urine not be distinctly green, this tint may be more clearly 
brought out by adding a few drops of acetic acid and 
boiling. Variations from the periods indicated above de- 
note a hyperacidity or a hypoacidity of the gastric juice 
according as the time of appearance of the reactions is 
lessened or increased. As the gut is digested only by 
the gastric juice 1 a non-appearance of either reaction 
would indicate an anachlorhydria. 

The writer has used these desmoid bags in a large num- 
ber of cases and has found them fairly reliable, giving re- 
Fig. 18. — Sahli s . J ' ° ° 

Desmoid bag. suits which have, in many cases, been confirmed by chemi- 
cal analysis. As these bags are not obtainable in the market, 
he has been forced to make them himself and has found that the technic of 
Sahli must be followed very closely, especially as regards the tying of the gut. 

Other methods, such as those of Dunham, Turck, and Einhorn, have been 
advocated, but possess no advantages over those outlined. 2 

IX. The Gastric Juice in Disease. 

(i). .Hyperchlorhydria. 

By hyperchlorhydria is meant the secretion of an abnormally acid gastric 
juice whose acidity is due to an excess of free hydrochloric acid. This secretion 
is much more marked during digestion, being less frequent on an empty stomach. 
Under these conditions we usually find an increased total acidity along with 
the increase of free hydrochloric acid. A condition which is characterized by a 
high total acidity with a very high amount of organic acid would not, of course, 
be considered in this connection. A hyperacidity or hyperchlorhydria exists 
when we have more than 0.2 per cent. (6o°) of free hydrochloric acid (see 
p. 69). 

This condition may be due to pathologic changes in the mucosa or to direct 
nervous influences. Cases of pure hyperchlorhydria are occasionally very 
stubborn and may be associated with almost any variety of abnormal gastric 
function. While we find hypermotility of the stomach in many cases of hyper- 

1 Gregersen (Arch. f. Verdauungskr., 1913, XIX, 43) advocates the use of Schmidt's test of 
administering 100 grams of very slightly cooked meat and the later study of the stools for con- 
nective-tissue fiber. 

2 See Friedrich, Berl. klin. Wchnschr., 1913, L, 32; also, Rehfuss, Am. Jour. Med. Sc, 
19 14, CXLVII, 848. 



GASTRIC CONTENTS. 89 

chlorhydria, we very frequently note a diminished motility due to spasm of the 
pylorus. This condition brings about a stagnation of the stomach contents 
and a consequent increase in the fermentative processes. The acidity in such 
cases may run as high as 200 or over and the digestive powers of the gastric 
juice, as regards protein substances, may be much increased, the carbohydrate 
digestion being correspondingly diminished. These facts point to the reason 
for the administration of an increased protein diet in such cases, the protein 
combining with the hydrochloric acid and thus taking a portion of the excess 
from the field of action. 

While this condition is not a distinct entity, yet we find many cases which 
come under the heading of idiopathic hyperchlorhydria and which are not 
associated with other pathologic conditions. Some of these cases are purely 
functional and clear up promptly under proper treatment, while others are of 
nervous origin and are remedied only when the etiologic factor is eliminated. 
In this latter type of hyperchlorhydria the degree of acidity varies with the 
nervous symptoms, giving rise to the term " heterochylia." 

(2). Hypersecretion (Gastrosuccorrhea). 

By hypersecretion is meant an excessive secretion of gastric juice which 
is out of proportion to the physiologic stimulus. This hypersecretion occurs 
even when no stimulus is present, is always pathological, and, according to 
Riegel, always produces pathological results. A hyper- or continuous secretion 
may be determined by finding a fairly large amount of gastric juice in the fast- 
ing stomach under conditions which rule out stenosis and stagnation. The 
stomach is washed out before the patient retires, the contents being withdrawn 
the following morning. If a quantity of highly acid fluid is obtained, a hyper- 
secretion is proven. The quantity taken from the fasting stomach should 
never be more than 100 c.c. This secretion, to be called a hypersecretion, must 
contain no food remnants, no sarcinae nor yeast cells, but should be distinctly 
acid. 

This condition is probably a functional neurosis, being constant or inter- 
mittent and a part of a general neurosis, a secretory neurosis, or the result of 
organic nervous disease, such as the gastric crises of tabes dorsalis. Reich- 
mann has reported cases of the periodic or intermittent type, during the inter- 
vals between the attack the digestion of the patient being normal. Such cases 
are known as Reichmann's disease. 

The chronic cases are of long duration and have a gradual onset. The 
patient complains of much discomfort, feeling of weight or depression in the 
stomach, pain during digestion, vomiting, especially at night, and a gastric 
contents with a large amount of free hydrochloric acid. Dilatation of the 
stomach sooner or later comes on as the result of spasm of the pylorus induced 
by the hyperacidity. In these dilated stomachs we find, of course, products 
of fermentation and many yeast cells and sarcinae. 

(3). Achylia Gastrica. 

This condition may arise either from a functional disturbance of the 
mucosa or a true atrophy of the mucosa. This latter state, known as atrophic 



90 DIAGNOSTIC METHODS. 

gastritis, may be the end stage of a chronic gastritis or the result of carcinoma. 
When this condition is not due to direct gastric disturbance, it is more fre- 
quently seen in connection with pernicious anemia, in which the general nutri- 
tion is very much below par. The local condition may not be suspected, as the 
hypermotility, so common in achylia gastrica, may prevent attention being drawn 
to the stomach. 

For a diagnosis of achylia, the test meal of Ewald gives very good results. 
Examination of the gastric contents shows that the food is little changed, the 
total acidity very low (i to 6°), no free hydrochloric acid, gastric ferments 
much diminished or entirely absent, and lactic acid only in small amounts. 
The motility of the stomach is usually little impaired, so that the retention of 
food is unusual. 1 

(4). Acute Gastritis. 

The stomach contents of acute gastritis shows a diminished total acidity, 
little or no free hydrochloric acid, organic acids relatively increased, much 
mucus and undigested food. This condition is usually brought on by direct 
irritation and is generally easily remedied by total abstinence. The material 
for chemical examination is usually obtained in these cases from the vomitus, 
as the passing of the stomach-tube is very rarely tolerated. 

(5). Chronic Gastritis. 

All grades of this condition may exist up to complete atrophy of the mucosa. 
Examination of the stomach contents shows practically no digestion of the food 
material, much mucus intimately mixed with the food particles, the secretion 
usually diminished, free hydrochloric acid diminished or absent, ferments 
much reduced, protein digestion small, starch digestion little affected, micro- 
scopic examination showing the presence of many epithelial cells and leuco- 
cytes. There are some cases of chronic gastritis in which a hyperacidity of the 
juice is evident, but these are rare, the usual finding being one of diminished 
acidity. The motility of the stomach in these cases is sometimes normal, some- 
times increased, or may be diminished. One of the most characteristic findings 
in this condition is the presence of a large amount of mucus containing either 
leucocytes or their nuclei and epithelial cells from the walls of the stomach. 
If there is little acid present in the gastric contents the mucus may swell up and 
appear greater in volume. As a general rule, it may be said that the mucus 
and the hydrochloric acid vary inversely as their amounts. 

(6). Nervous Dyspepsia. 

This condition of nervous dyspepsia is part of a general neurosis and may 
show no characteristic findings in the stomach contents. The degree of 
acidity may range from a normal to either a hyper- or a hypoacidity, while the 
amount of ferments present will not usually vary. The findings in nervous 
dyspepsia are not at all constant, varying at different examinations. We have, 
therefore, more or less distinct methods of differentiation between this con- 

1 Disque (Arch, f . Verdauungsk'r., 1914, XX, 366); Orloff (Russk Vrach., 1914, XIII, 1006) 
believes there eventually occurs a vicarious increased secretion of trypsin in this condition. 
See, also, Rehfuss, Am. Jour. Med. Sc, 1915, CL, 72. 



GASTRIC CONTENTS. 9 1 

dition and chronic gastritis. In the first place, the acidity of chronic gastritis 
remains constant for several examinations while that of nervous dyspepsia is 
variable. The ferments are diminished in cases of gastritis, while they are 
normal in nervous dyspepsia. Much mucus is found in gastritis while little 
or none is present in dyspepsia. The cases of nervous dyspepsia are partially 
associated with distinct errors in eating. The influence of nervous conditions 
over gastric function has been very well expressed by Emerson when he says, 
"a neurasthenic will often worry his subliminal gastric sensations into the sphere 
of consciousness." 

(7). Ulcer of the Stomach. 

The diagnosis of ulcer of the stomach depends, to a large extent, upon the 
clinical symptoms of the disease rather than upon the examination of the 
stomach contents. 1 While the symptoms of this disease, increasing dyspepsia, 
pain, vomiting, blood in the vomitus, and hyperacidity of the vomitus, are well- 
known, the stomach-tube should rarely ever be "used in obtaining the contents 
of the stomach, owing to the danger of perforation in such cases. 2 

The vomitus of such cases is usually ejected from one to three hours after a 
meal and contains well-digested food. Blood may or may not be present and 
may be either fresh red blood or dark in color from the formation of hematin. 
The total acidity of the gastric contents is usually increased, hydrochloric acid 
constituting a large part of this total acidity. A single examination of the 
gastric contents will rarely determine anything about an ulcer, so that repeated 
examinations of the vomitus must be made to obtain a general idea of the 
acidity. In these cases blood is usually present in the feces and may be de- 
tected as outlined later. 3 When an ulcer is complicated by a beginning car- 
cinoma, we may find all types of variation in the acidity of the stomach contents. 

(8). Carcinoma of the Stomach. 

In no other condition of the stomach is an absolutely certain early diagnosis 
to be so much desired as in carcinoma of the stomach. The chemical features 
of the gastric juice in this condition may be very suggestive or may be negative. 
The clinical history of the case along with the age of the patient are probably 
of more importance in making a diagnosis of carcinoma than are variations in 
the chemical composition of the juice. The local symptoms of carcinoma of the 
stomach are sometimes as variable as are the changes in the gastric juice, so that 
every possible point in diagnosis should be taken advantage of, if for no other 
purpose than to exclude this condition. 4 

Perhaps the most important sign of carcinoma is the absence of free hydro- 
chloric acid. Although this condition is present in about 85 per cent, of cases, 

1 See Verbrycke, Am. Jour. Med. Sc, 1913, CXLVI, 742. 

2 Rosenow (Jour. Am. Med. Assn., 1913, LXI, 1947) has shown the close relationship of 
streptococcic invasion to gastric ulcer. See, also, Rosenow and Sanford, Jour. Infect. Dis., 
1915, XVII, 219. 

3 See Lichty, Am. Jour. Med. Sc, 1914, CXLVIII, 680; Smith, Jour. Med. Research, 1914, 
XXX, 147; Smithies, Arch. Diagnosis, 1915, VIII, 147; Interstate Med. Jour., 1915, XXII, 207; 
Boas, Arch. f. Verdauungskr., i9i5,XXI, 94; Sippy, Jour. Am. Med. Assn., 1915, LXIV, 1625; 
Mayo, Ibid., 191 5, LXV, 1069; Cheney, Ibid., 1227. 

4 See Schutz, Wien. klin. Wchnschr., 19 13, XXVI, 1053. 



92 DIAGNOSTIC METHODS. 

it cannot always be traceable to carcinoma, as it may occur in atrophic gastritis 
and advanced chronic gastritis. This lack of free hydrochloric acid is due to 
the union of this acid with some body which in itself does not show an alkaline 
reaction. Von de Velden suggests that the secretion from the cancer is the 
active agent in neutralizing the hydrochloric acid. Moreover, the products of 
protein digestion might have some power in neutralizing the acid, Emerson 
having shown that hexone bases are present as a result of the action upon the 
protein of a ferment 1 derived from the tumor itself. Certainly, in cases of 
carcinoma the total nitrogen of the stomach contents is much increased, so that 
such bodies are probably a very great factor in the diminution of the hydro- 
chloric acid. This reduction in hydrochloric acid is also influenced by 
changes in the mucosa to such an extent that the active secretion is diminished. 
The failure of free hydrochloric acid is usually a very early symptom, but it 
must be remembered that hydrochloric acid may be present in normal amounts 
or even in increased amounts when the carcinoma is small and occupies the 
pyloric region or when this growth develops on the base of an old ulcer. 2 The 
acidity may vary markedly from day to day, sometimes showing no free hydro- 
chloric acid and sometimes a considerable amount. This variation is of con- 
siderable practical importance. In cases of carcinoma of the esophagus the 
disappearance of hydrochloric acid from the gastric contents seems necessarily 
to be the result of the secretion of the tumor neutralizing the gastric juice. Along 
with this diminution in free hydrochloric acid the total acidity is also diminished. 3 
The presence of an increased amount of lactic acid is a very valuable sign 
in cancer. 4 About 90 per cent, of patients show the presence of lactic acid when 
there is no free hydrochloric acid, but when there is a large amount of combined 
HC1 pointing to a free secretion of this acid. Lactic acid may not be present 
in cases of carcinoma, especially those in which the growth is upon the base of 
an old ulcer, or it may be present in conditions other than carcinoma, such as 
chronic gastritis, associated with atrophy of the mucosa and dilatation of the 
stomach, especially when a benign stenosis of the pylorus exists. It must be 
stated, however, that we usually find an increased secretion of hydrochloric 
acid in cases of simple benign pyloric stenosis, so that lactic acid cannot be 
formed in the presence of this increased hydrochloric acid. According to 
Riegel, the chief cause of the lactic acid formation is the combination of motor 
insufficiency with a hypoacidity associated with a diminished secretion, both 
of acids and ferments. This diminution in the amount of ferments is in no way 
specific for cancer, as it is in reality due to the chronic gastritis which is set up 
by the tumor. As lactic acid cannot be formed in the presence of an appre- 
ciable amount of free HC1, we do not always find after a test meal a large excess 
of this acid, especially in those cases in which considerable combined HC1 is 

1 Kriloff (Med. Obozr., 19 13, LXXIX, 1) believes trypsin is always present in cancer, 
especially of the pylorus. See, also, Halpern, Mitt. a. d. Grenzgeb. der Med. u. Chir.. 1914, 
XXVIII,' 709. 

2 See Levy, Beitr. z. klin. Chir., 1914, XCIII, 696; Ochsner, Jour. Am. Med. Assn., 1915, 
LXV, 1073. 

3 See Friedenwald, Am. Jour. Med. Sc, 1914, CXLYIII, 660; Smithies, Jour. Am. Med. 
Assn., 1914, LXIII, 1839; Ibid., 1915, LXIV, 643. 

4 See Schryver and Singer, Quart. Jour. Med., 1913, VI, 71 and 309. 



GASTRIC CONTENTS. 93 

present. It seems advisable, therefore, in testing for the presence of lactic 
acid, to examine the contents of the fasting stomach in the morning after it 
has been well washed out the preceding evening. Whether we are to assume 
that lactic acid is formed by the action of the organisms in the stomach upon 
food material, whether it be a normal product of digestion, or whether it is a 
product of the activity of a ferment derived from the tumor, must be left for 
future investigation. 

The vomited material or the material obtained by washing out the stomach 
shows very little digestion of the protein elements, while the carbohydrates are 
well hydrolyzed. The amount of material in the stomach will be large or small 
depending upon the degree of stenosis, so that it is nothing unusual to obtain 
several pints of material containing undigested protein residue. Microscopic 
examination of this material may show cellular masses washed off from the 
tumor or may show these fragments embedded in masses of blood. 1 Blood 
is a usual finding in cases of carcinoma and may be detected by the methods dis- 
cussed under Feces. Sarcinse and yeasts are rare, the former occurring more 
frequently in cases of marked dilatation. The Boas-Oppler bacilli are more 
or less constant findings in carcinoma, occurring in about 90 per cent, of the 
cases and only very rarely in any other condition. 

Salomon's Test. 

This test is based upon the fact that albumin is secreted from the car- 
cinoma itself and passes into the gastric contents. The patient is placed upon 
an absolutely protein-free diet for 24 hours and the stomach carefully washed 
out at the end of this time with 400 c.c. of physiological salt solution. A few 
hours thereafter the contents of the stomach are removed and the remaining 
material washed out with 400 c.c. of physiological salt solution. The total 
nitrogen and the albumin are then estimated, the former by the Kjeldahl 
method and the latter by the Esbach method (see Urine). The nitrogen 
ranges from 10 to 70 mg. per 100 c.c. in cases of carcinoma, while in other 
conditions it varies from o to 16 mg. per 100 c.c. The Esbach reaction gives a 
distinctly appreciable precipitate for albumin, anything over 0.5 parts per 
1000 being considered indicative of carcinoma. While this test is not infallible, 
yet the writer has found it present in so many cases of carcinoma that he is 
inclined to make a very strong presumptive diagnosis on the basis of this test. 2 

Neubauer and Fischer's Test. 

These investigators, 3 working in M tiller's clinic, have taken advantage 
of the well-known fact that normal peptic digestion does not proceed to the 
formation of amino-acids but stops at the peptone or peptid stage. Further, it 
has been shown by Emerson, Fischer and others that digestion of protein goes 
somewhat further in carcinomatous stomachs than in normal ones, owing to the 
fact that carcinomatous tissue, as well as various body tissues, contains pro- 
teolytic ferments capable of producing more complete hydrolysis than does 

1 See Simon and Caussade, Presse med., 1914, XXII, 265; Loeper and Binet, Arch, des 
Mai. de l'App. digestif, 1914, VIII, 181; Caussade, Rev. de Med., 1914, XXXIV, 42S. 

2 See Hohlbaum, Arch. f. klin. Chir., 19 14, CIV, 1069. 

3 Deutsch. Arch. f. klin. Med., 1909, XCVII, 499. 



94 DIAGNOSTIC METHODS. 

pepsin (see Lewin 1 ). Neubauer and Fischer, following the investigations of 
Erdmann and Winternitz, Glaessner, and Volhard, use this hydrolysis of 
peptids by such proteolytic ferments for diagnostic purposes. Their method is 
as follows: The contents of the stomach are withdrawn one-half to three- 
fourth hour after an Ewald meal; ioc.c. of the filtered material are mixed with 
a little glycyl-tryptophan 2 and a little toluol is added to prevent bacterial action. 
The mixture is placed in the incubator at 37 C. for 24 hours. At the end of 
this period, withdraw 2 or 3 c.c. of the mixture from beneath the toluol layer, 
place this in a test-tube, add a few drops of 3 per cent, acetic acid and then 
allow the fumes of bromine' to pass into the tube from an open bottle of bromine. 
This part of the technic must be carried out very carefully as an excess of 
bromine will obscure and even destroy the reaction. After shaking the tube 
a positive reaction is shown by the appearance of a rose-red or intense red color. 
If no red color appears, carefully add more bromine vapor and shake. This 
addition may be continued until an excess of bromine is evidenced by a light 
yellow color of the mixture. 

These workers have shown that errors may arise in this test due to the 
presence of (1) tryptophan in the gastric contents, (2) peptid-splitting bacteria, 
(3) trypsin or pancreatic juice, and (4) blood. To do away with these sources 
of error they advise the rejection of gastric contents showing any of these 
substances. Filtering the contents removes the peptid-splitting bacteria 
to some extent while the addition of toluol prevents their development. Tryp- 
tophan, as such, may be tested for as usual, while the presence of pancreatic 
juice may be assumed if bile be present. Blood is shown by tests outlined under 
Feces. Warfield 3 has recently shown another fallacy of this test, which may 
render the test of little clinical value. He has found that the saliva of many 
patients is capable of decomposing glycyl-tryptophan into its constituent 
elements. He concludes, therefore, that swallowed alkaline saliva, when mixed 
with neutral or faintly acid (not over o. 05 per cent. HC1) gastric contents, may 
introduce sufficient error to account for the discordant results obtained. Koelker 4 
has found a di- and tripeptid-splitting ferment in the saliva. Lyle and Kober 5 
believe that a negative result with this test is of far greater value than is a 
positive one. 

Weinstein 6 believes that the presence of tryptophan in the gastric con- 
tents, as withdrawn, is more positive as a test for carcinoma than is the glycyl- 
tryptophan test, providing intestinal fluid has not regurgitated into the stomach. 
He has confirmed the findings of Warfield and asserts that this latter fallacy 
can not be applied to his "tryptophan test" as the salivary proteolytic action 
is not such as to produce tryptophan from protein in non-cancerous gastric 

1 Ergebn. d. inn. Med., 1908, II, 212. 

2 Glycyl-tryptophan, as prepared for the test, is a clear solution. It is furnished by 
Kalle & Co., Biebrich a. Rhein, under the name of "Fermentdiagnostikum." It comes in 
small bottles which are used directly for the test by simply adding the 10 c.c. of filtered gas- 
tric contents and incubating for 24 hours. 

3 Bull. Johns Hopkins Hosp., 19 11, XXII, 150. 
4 Ztschr. f. physiol. Chem., 1911, LXXVI, 27. 

5 New York Med. Jour., 1910, XCI, 1151. 

6 Jour. Am. Med. Assn., 1910, LV, 1085; Ibid., 1911, LVII, 1420. 



GASTRIC CONTENTS. 95 

contents. He, therefore, suggests the following modification. A portion 
of the gastric contents is withdrawn 4 or 5 hours after a full dinner and the 
filtered contents tested directly for tryptophan after acidifying the filtrate 
with acetic acid. If no reaction appears, incubate for 24 hours and repeat the 
test. Owing to the large number of possible fallacies and the discordant 
results obtained with this test, it seems probable that it, like so many others, 
must be regarded as of little diagnostic value. 1 

Friedman and Hamburger (Arch. Int. Med., 19 13, XII, 346) believe that 
the proteolytic cleavage of stomach contents is due, in most instances, to regurgi- 
tated trypsin, although leucocytes and bacteria play some role. They advocate a 
combined method of proteolysis and peptolysis, which they believe preferable 
to other tests advanced. A high peptolysis with low proteolysis speaks for 
carcinoma; while a high peptolysis and high proteolysis speaks against car- 
cinoma. This test promises to be a reliable one but confirmatory work must 
establish its status. 

Wolff and Junghans* Test. 

These workers 2 have originated a test for the differentiation of benign and 
malignant achylias, which test seems to have considerable diagnostic value if 
properly controlled and interpreted. The test is based on the following ob- 
servations. Normal aspirated test meals show a relatively large amount of 
soluble albumin, which arises from the digestion of the protein of the test meal 
by the gastric ferments in the presence of free hydrochloric acid. This albumin 
increases in amount in proportion to the time which it remains in contact with 
the normal gastric juice. In conditions associated with achylia or with marked 
diminution of hydrochloric acid in the stomach, it would be reasonable to sup- 
pose that little or no protein digestion would occur. This was found to be the 
case in benign achylias but not true in the malignant types. In these latter 
cases the soluble albumin (probably a proteose) was found to be much increased. 
This albumin may arise (1) from protein retention from a previous meal, due to 
stasis, lack of motor tone, or true obstruction, as shown by Clarke and Rehfuss; 
(2) from mixing of the test meal with so-called "cancer juice" exuded from the 
malignant growth; (3) from the presence of blood or pus arising from ulcerative 
lesions; (4) from the presence of swallowed saliva or, especially, sputum; and 
(5) from the action of a specific peptid-splitting ferment arising from the malig- 
nant growth and capable of splitting the protein into lower groupings of amino- 
acids. This latter source is the most important, although the others must be 
excluded before proper interpretation is possible. In other words, one must be 

1 In this connection see Taylor and Hall, Jour. Path, and Bacterid., 1912, XVII, 121; 
Sanford and Rosenbloom, Arch. Int., Med., 1912, IX, 445; Smithies, Ibid., 1912, X, 357 and 
521; Jacque and Woodyatt, Ibid., 560; Hamburger, Jour. Am. Med. Assn., 1912, LIX, 847; 
and Smithies, Ibid., 1913, LXI, 1793. 

2 Wolff and Junghans, Berl. kJin. Wchnschr., 1911, XLVIII, 978; Wolff, Magen und Darm- 
krankheiten, Berlin, 1912, 217. See, also, Thiele, Berl. klin. Wchnschr., 1912, XLIX, 544; 
Einstein, Med. Klin., 19 12, VIII, 484; Roubitschek and Weiser, Arch. f. Verdauungskr., 
1913, XIX (Erganz. Bd.), 146; Rolph, Med. Record, 1913, LXXXIII, 848; Smithies, Am. 
Jour. Med. Sc, 19T4, CXLVII, 713; Trallero, Deutsch. med. W T chnschr., 1914, XL, 1428; 
Kahn and Jacobowitz, Biochem. Bull., 1915, IV, 214; Lenskaia, Russk. Vrach., 1915, XIV, 
267; Katznelson, Ibid., 292; Clarke and Rehfuss, Jour. Am. Med. Assn., 1915, LXIV, 1737. 



90 DIAGNOSTIC METHODS. 

sure (i) that the stomach is free from remnants of previous protein meals, as in 
some cases this gastric residuum may be sufficient to give erroneous results; 1 (2) 
that blood and pus are absent, at least in more than traces; and (3) that sputum 
especially is not swallowed during the period of retention of the test meal, as 
some types of sputum are relatively rich in soluble albumins. 

While, theoretically, typical cases of advanced gastric carcinoma, at least, 
should show little or no free hydrochloric acid, it should be remembered that 
such is actually not the case, so that this test applied to cases in which free 
hydrochloric acid is present to an appreciable extent may, of course, give 
high albumin values due to ordinary gastric digestion. However, the studies of 
Clarke and Rehfuss, by means of their fractional method of gastric examination, 
show that the curve of protein digestion under normal conditions follows the 
acid curve rather sharply, while in malignant states the protein curve diverges 
from the acid curve and, as digestion progresses, this disproportion increases 
until there is a marked separation of the curves. From these facts, these workers 
argue that the fractional study of both the curves of gastric acidity and protein 
digestion should be simultaneously studied before the proper interpretation can 
be reached. 

Technic. 

In order to avoid the possibility of retention of protein residues in the 
stomach, the patient is allowed a light meal with very little protein on the evening 
before the examination is to be made. A light cathartic is administered the 
same evening. The next morning administer an Ewald test breakfast. With- 
draw this meal within 45 to 60 minutes. Filter the contents through filter-paper 
until clear. 

While filtration is proceeding, arrange a series of six clean test-tubes in a 
rack, numbering the tubes serially. By means of a 1 c.c. pipet, which is grad- 
uated in divisions of 1/100 c.c, place in the respective tubes 1 c.c, 0.5 cc, 
0.25 c.c, 0.1 c.c, 0.05 c.c, and 0.025 c - c - Now make up the volume in each 
tube to 10 c.c by the addition of distilled water from a finely graduated pipet. 
The dilutions in these tubes will be, then, 1 to 10, 1 to 20, 1 to 40, 1 to 100, 1 to 
200, and 1 to 400. Intermediate and extra dilutions may be made if it is desired 
to work the reaction down to a much finer point. Mix the contents of the tubes 
thoroughly by shaking and inverting the tubes. 

Overlay the contents of each tube with 1 cc of the reagent to precipitate 
the albumin and note the appearance of a white ring at the point of contact of 
the fluid and reagent in the various tubes. A black background will bring out 
these rings more distinctly. 

Wolff's reagent is as follows: 

Phosphotungstic acid „ 0.3 gram. 

Concentrated Hydrochloric Acid 1 . o cc 

Alcohol (96 per cent.) 20 . o c.c. 

Distilled water ad 200 . o cc. 

1 See Rehfuss, Bergeim and Hawk, Jour. Am. Med. Assn., 1914, LXIII, 11. 



GASTRIC CONTENTS. 97 

Interpretation. 

Normally a distinct ring of albumin will be seen at dilutions of i to 10, i to 
20 and, occasionally, at 1 to 40. This reaction, which is called negative at these 
dilutions, may even reach, in very rare cases, as high as 1 to 200 due to various 
factors spoken of above, especially to the presence of blood and retention of 
protein residues. The importance of excluding extrinsic sources of albumin is 
thus evident. Generally speaking, however, the diagnosis of cancer becomes 
more probable the higher the dilution at which the albumin precipitation occurs, 
although cancer cannot be excluded in some cases when the precipitation does 
not appear in the last tubes. A precipitation unit of 100 or over speaks rather 
markedly for a diagnosis of cancer, presumption becoming almost a relative 
certainty when the unit is 200 to 400. This implies, of course, that all the points 
mentioned above have been taken into consideration. Smithies regards a 
reaction at a dilution of 100 as doubtful. He states that this test a was a more 
constant finding in gastric extracts than were absent free hydrochloric acid, the 
presence of lactic acid and the glycyltryptophan test. It was rather more 
constant than tests for occult blood and the demonstration of motor inefficiency. 
It was not so consistent in its manifestation as the demonstration of organisms of 
the Boas-Oppler group or the increase in the formol index." 

It will be seen, therefore, that this test, of positive precipitation of albumin 
in dilutions of the gastric extract from 1 to 200 up, is of considerable value in the 
differentiation of the benign and malignant achylias or achlorhydrias, especially 
if the former are not associated with motor insufficiency. In cases of ulcer, 
especially if pyloric stenosis obtains, positive Wolff- Junghans' tests are not in- 
frequent, indicating that symptomatology, gastric acidity and other findings 
must be taken into consideration. Like many other tests for cancer, this must 
not be relied upon to settle the diagnosis, to the exclusion of other laboratory 
and clinical data. With proper technic and care in excluding extrinsic factors, 
this test has certainly more than a presumptive value, giving definitely positive 
findings in fully 80 per cent, of disputed cases. 



BIBLIOGRAPHY. 

1. Boas. Die Erkrankungen des Magens. Berlin, 1906. 

2. Billings. Diseases of the Digestive System. New York, 1906. 

3. Debove, Achard, et Castaigne. Manuel des Maladies du Tube Digestif. 

Paris, 1907. 

4. Ewald. Die Erkrankungen des Magens. Berlin, 1900. 

5. Harley and Goodbody. The Chemical Investigation of Gastric and 

Intestinal Diseases. London, 1906. 

6. Hemmeter. Diseases of the Stomach. Philadelphia, 1906. 

7. Loeper. Manuel des Maladies du Tube Digestif. Paris, 1907. 

8. Lyon. Diagnostic et Traitement des Maladies de L'Estomac. Paris, 1909. 

9. Reed. Diseases of the Stomach and Intestines. New York, 1907. 

10. Riegel. Die Erkrankungen des Magens. Wien, 1903. 

11. Roeser. La Chimie Alimentaire. Paris, 1906. 

12. Sigaud. Traite clinique de la digestion. Paris, 1907. 

7 



98 DIAGNOSTIC METHODS. 

13. Smithies and Ochsner. Gastric Cancer. Philadelphia, 19 15. 

14. Stockton. Diseases of the Stomach and Their Relation to Other Diseases. 

New York, 19 14. 

15. Zunz. Methodik der Mageninhaltuntersuchung, Handb. d. Biochem. Arbeits- 

Methoden, 19 14, VIII, 44. 

16. Zweig. Diagnose und Therapie der Magen- und Darmkrankheiten. Wien, 



CHAPTER IV. 
THE FECES. 

I. General Considerations. 

The feces are composed of substances of different origin, which may be 
divided as follows: (i) Food remnants, either undigestible constituents or 
digestible but unabsorbed elements; (2) secretions of the alimentary tract; (3) 
decomposition products and bacteria; (4) formed and unformed elements 
derived from the intestinal wall, and (5) foreign bodies, such as hair, wood 
£ber, parasites, parasitic ova, and enteroliths. It is not possible to draw a 
sharp line between a truly physiologic and pathologic composition of the feces. 
In each individual case this division will depend upon a number of factors, 
among which may be mentioned composition of the food, method of taking the 
food, individual functional capacity of the intestines, frequency of the bowel 
movements, and general systemic conditions. The condition of the food 
remnants will give much information regarding the functional capacity of the 
bowel. Under normal conditions, about one-third of the dry substance of the 
feces arises from the bacteria, this amount increasing under certain patho- 
logic conditions which will be later discussed. As products of bacterial 
activity we observe the formation of hydrobilirubin from bilirubin as well as 
the reduction of certain medicaments and the rare formation of certain diamins, 
which will be treated later. This bacterial activity will vary much under 
pathologic conditions and may have much to do with the symptomatology of 
the case investigated. 1 The ordinary decomposition products are derived from 
the carbohydrates and proteins of the food; from the former are produced, by 
fermentation, volatile fatty acids, lactic acid, succinic acid, alcohol, carbon- 
dioxid, hydrogen, and methane, while from the latter are formed by putre- 
faction indol, skatol, phenol, ammonia, and hydrogen sulphid. The fats are 
decomposed only to a very slight extent. The normal products of digestion 
in the intestinal tract as well as the factors bringing about these changes will be 
discussed later. 2 

Normal Feces. 

For proper comparisons between the feces of various individuals the food 
must be the same. Starvation feces, meat feces, and milk feces are typical 
types, but are not normal in the strict sense. For comparison, however, an 
arbitrary norm must be established in order to judge of slight variations which 
have to do with special differences in utilization of food and which are not 
observable by the eye except under certain conditions (as, for instance, fatty 

1 See von Noorden, Jour. Am. Med. Assn., 1913, LX, 101. 

2 See Sanford, Wisconsin Med. Jour., 1914, XII, 281; also, McClanahan and Moore, Am. 
Jour. Med. Sc, 1915, CXLIX, 815. 

99 



IOO 



DIAGNOSTIC METHODS. 



stools). Through the work of Praussnitz and of Schmidt and Strasburger a 
new field for study of normal and abnormal feces has been opened up. 

A normal feces should be one which consists almost entirely of remnants 
of the digestive juices and intestinal secretion arising from a purely digestible, 
properly prepared, and assimilable food. It contains approximately 8.6 per 
cent, of nitrogen, 16 per cent, of ether extract, and 15 per cent, of ash calculated 
on the dry basis. Any appreciable variation from this composition would 
indicate a diminution of the functional activity of the bowel. In the investi- 
gation of pathological cases this composition is scarcely to be assumed, as the 
diet is more or less restricted, the appetite is capricious, the intestinal activity 
is variable, and the feces are, therefore, different from those of the healthy. 
It must not be understood from this that a feces showing the above composition 







1£y0Ih %3m£'i'J' k - 

Fig. 19. — Normal Feces. (Landois.) 
a, Muscle fibers; b, tendon; c, epithelial cells; d, leucocytes, e-i, various forms of plant- 
cells, among which are large numbers of bacteria; between h and b are yeast-cells; 
k y ammonium-magnesium phosphate. 

is necessarily normal in other respects. One may not rely entirely on the 
chemical composition, but must largely consider the macroscopic and micro- 
scopic findings. In order that we may have a sound basis upon which to 
judge of intestinal activity it seems wise to have some sort of test diet which 
may be given to suspected cases. 

Diet of Schmidt and Strasburger. 

This diet 1 is so selected that it can be used by the healthy as well as by 
those with intestinal trouble; its amount is sufficient to satisfy the maximal 
calorie requirement of the individual while at rest; it contains the three chief 
groups of food material in definite relation to each other; is as free as possible 
from remnant-leaving food, and can be easily obtained and prepared. The 
starchy food present is of the amount and kind which have been shown most 
favorable for the prevention of excessive fermentation in the bowel. The 
daily diet is as follows: One and five-tenths liters of milk, 100 grams of 
zwieback, two eggs, 50 grams of butter, 125 grams of beef (raw weight), 190 
grams cooked potato, and a gruel of 80 grams of oatmeal. This is distributed 

1 Die Faeces des Menschen, Berlin, 191 5. 






THE FECES. IOI 

through the day as may best suit the patient. This diet contains 102 grams 
of protein, in grams of fat, and 191 grams of carbohydrate, yielding 2,234 
raw calories. In cases which show a diarrhea, due to the milk, one may sub- 
stitute instead of 1/2 liter of milk the same amount of cocoa made from 20 
grams of cocoa powder, 10 grams of sugar, 100 grams of milk, and 400 grams 
of water. Small variations in the amount of milk, sugar, butter, and even of 
eggs may be permitted, but the outline, as regards meat, zwieback, potato, 
and gruel should be rigidly adhered to. 

This diet should be administered for three days or longer if necessary 
to obtain a stool which comes from it. In order to judge of the first appearance 
of the stool from this diet, the patient should be given a capsule containing 0.3 
gram of powdered carmin, both preceding and following the diet. 1 Instead of 
carmin, one may use cork, charcoal, or silicic acid. 

Folin's Diet. 

This diet 2 is especially serviceable in case one wishes to follow the metabo- 
lism in any special case. Its easy application and its fairly constant values for 
nitrogen, phosphorus, chlorin, and sulphur make it invaluable. 

The standard diet, which is given to the patient daily for several days is 
as follows: 

Whole milk, 500 c.c. 

Cream (18 to 22 per cent, fat), 300 c.c. 

Eggs (whole), 450 gm. 

Horlick's malted milk, 200 gm. 

Sugar, 20 gm. 

Sodium chlorid, 6 gm. 

Water, 2,100 c.c. 

This diet contains approximately 119 gm. of protein, 148 gm. of fat, and 
225 gm. of carbohydrate, yielding 2787 raw calories. The intake of one day is 
nitrogen 18.9 gm., 5.9 gm. of P 2 5 , 3.8 gm. of S0 3 , and 6.2 gm. of CI. It is 
not to be compared with that of Schmidt and Strasburger for estimating the 
intestinal activity, but is more reliable if metabolism relations are to be studied. 

The normal motility of the feces is from 6 to 20 hours, while on a milk 
diet it may vary from 36 to 48 hours. This factor may be of importance in 
judging of intestinal obstruction. The isolation of a stool under a special diet 
is of great importance, as the success of an investigation will depend upon this. 

Obtaining Intestinal Juice. 

Boas has shown that it is often possible, by the use of the stomach, tube, 
to obtain sufficient intestinal secretion to permit of examination. His technic 
is, however, rather difficult, is trying to the patient and is often unsatisfactory. 
Boldyreff, Volhard, Faubel and, more recently, Koziczkowsky 3 have shown 

1 See Basch, Jour. Am. Med. Assn., 1913, LXI, 1295; Strauss, Arch. f. Verdauungskr., 
1914, XX, 299. 

2 Jour. Physiol., 1905, XIII, 45. 

3 Ztschr. f. klin. Med., 1909, LXVIII, 261. 



102 DIAGNOSTIC METHODS. 

that the intestinal juice may easily be obtained by the following method. The 
patient is allowed to take 150 c.c. of pure olive oil when the stomach is empty, 
preferably in the morning. At the end of three-fourth hour, during which time 
the patient must remain quiet in a sitting posture, the gastric contents are removed 
by means of a stomach tube. The material, obtained by means of this oil 
test-breakfast, 1 readily separates into two layers, an upper of oil and a lower of 
the mixed gastric and intestinal secretions. This may then be tested for the 
various ferments in the usual ways. 

Trypsin is best detected by the method of Gross. 2 Either the fluid 
obtained as above or a portion of the feces, rubbed up with three times its 
amount of a 1 to 1,000 Na 2 C0 3 solution and filtered, may be used for the test. 
100 c.c. of a 0.5 per thousand solution of casein in 1 to 1,000 Na 2 C0 3 solution 
are treated with 10 c.c. of fluid mentioned above and the mixture placed in the 
incubator for eight to twelve hours. At the end of this period the addition of a 
few drops of a dilute (1 per cent) acetic acid should produce no precipitate in 
case digestion is complete, that is if trypsin is present. 

Functions of the Intestinal Juices. 

The pancreatic juice as excreted into the intestine is an alkaline fluid con- 
taining three ferments, trypsin which hydrolyzes protein, amylopsin which 
acts upon the carbohydrates, and lipase (steapsin) which aids in the digestion 
of fat. The trypsin appears to be excreted in the form of a zymogen which 
is activated by a second ferment, enterokinase, derived from the intestinal 
mucosa, while the lipase and amylopsin are active when secreted. It has been 
shown by Pawlow that the passage of free hydrochloric acid into the duodenum 
is a direct stimulant to the excretion of pancreatic juice. Bayliss and Starling 
believe, however, that the stimulus to the pancreatic secretion is not the free acid 
but is a ferment, secretin, which is formed by the action of the hydrochloric 
acid upon the intestinal mucosa. While the trypsin acts upon protein bodies, 
splitting them through various stages into the ultimate products, amino-acids 
and hexone bases, there is a second ferment, erepsin, discovered in the intestinal 
mucosa by Cohnheim, which acts upon the intermediate splitting products of pro- 
tein, such as the albumoses and pepton, carrying this conversion to the same 
lengths as does trypsin. 3 It is interesting to note that we find in the intestinal 
juice of the infant a ferment, lactase, which hydrolyzes lactose into the simpler 
saccharids. 

The presence of the bile, which reaches the duodenum through the common 
duct, is necessary for the proper digestion and absorption of the fatty sub- 
stances of the food. Variations in this constituent may reflexly cause disorders 
in the secretion of the stomach, one finding very frequently a hyperacidity 
associated with obstructive jaundice. Whether the bile maybe assumed to have 
a disinfecting power must be left for the future. Were this influence actually 

1 See Matko, Med. Klin., 1913, IX, 1546 and 1587. 

2 Deutsch. med. Wchnschr., 190Q, XXXV, 706. 

3 See Schlecht and Wittmund, Deutsch. Arch. f. klin. Med., 1912, CVI, 517; Friedmann, 
Med. Record, 1912, LXXXI, 355; Abderhalden, Ztschr. f. physiol. Chem., 1912, LXXVIII, 
344- 



THE FECES. IO3 

present, it is hard to understand, why, in cases of long-standing exclusion of bile, 
we do not find catarrhal conditions as we do in cases of marked putrefactive 
processes in the bowel. 

Estimation of Intestinal Digestion. 

A careful chemical examination of the feces, coupled with a macroscopic 
and microscopic investigation, will give much information regarding the degree 
of intestinal digestion^ and absorption. However, such methods are time- 
consuming and not easily performed by the general practitioner. For this 
reason Sahli has introduced a method, similar to his stomach method, of 
investigating such activity. He employs glutoid capsules, which are made 
from gelatin hardened with formaldehyd. These capsules either do not 
dissolve in the gastric juice at all or only after considerable time, although they 
are quickly soluble in the intestinal juice. In these capsules is placed material 
which will not diffuse through the capsule wall and whose absorption may be 
studied from an examination of the saliva or urine. Sahli uses either iodoform 
or salol. In the former case 0.15 gram of iodoform is placed in a glutoid 
capsule, and given with an Ewald test meal. Under the best conditions (normal 
gastric motility, normal intestinal digestion, and normal intestinal absorption) 
the iodin reaction may be expected to appear, according to Sahli, in the saliva 
within four to six hours; that is, within one and one-fourth hours after the 
capsule has been dissolved by the pancreatic juice. Instead of iodoform salol 
may be used, being given in the amount of one-half gram along with an Ewald 
test meal. The reaction for salicyluric acid may be obtained in the urine 
within one and one-half hours after the capsule has been taken. 

Sahli gives the following results obtained by the use of this capsule: When 
the stomach contains neither free hydrochloric acid nor pepsin, the reaction is 
not delayed so long as gastric motility is good. In cases of diarrhea due only 
to an increased peristalsis without any marked disturbance of intestinal diges- 
tion, the reaction is either normal or even somewhat hastened. In other types 
of diarrhea characterized by an involvement of the intestinal chemistry or in- 
testinal absorption, the reaction is either absent or distinctly delayed or the 
capsules may be found undigested in the feces. This method also aids in 
differentiating an icterus due to occlusion of the ductus choledochus at its 
point of entrance into the intestine, in which case the digestion of the capsule 
may be interfered with, from one where the obstruction to the bile flow is higher 
up near the liver. This test may be of some presumptive evidence in the 
diagnosis of pancreatic carcinoma, although all cases of pancreatic carcinoma 
do not necessarily occlude the duct, in which case a positive result would obtain. 
Galli reports a case of carcinoma of the pancreas in which the glutoid capsule 
was dissolved, although no pancreatic juice was present in the bowel. 

II. Macroscopic Examination. 

(1). Method. 

The macroscopic examination of the feces embraces not only the study of 
the physical characteristics, but also a recognition of various normal and 



104 



DIAGNOSTIC METHODS. 



abnormal substances. It is convenient, when examining the feces, to employ 

some form of a washing apparatus to separate the coarser from the finer 

particles. 

Boas has introduced a special sieve for such work which, however, does not 

have much advantage over the ordinary flour-sifter which Einhorn advises. 

Strauss' method of washing the feces by a 
current coming from below seems to be much 
the best of any of these types of apparatus. 
In the absence of any other equipment, an 
ordinary house-hold sieve of various-sized 
mesh will answer the purpose. Small 
amounts of mucus or connective tissue frag- 
ments may be recognized by rubbing up a 
portion of the stool in a mortar with a little 
water, when these substances will float upon 
the surface. In some cases it may even be 
advisable to place the feces in tall glass jars 
in which the stool is mixed with water and 
allowed to arrange itself in layers. 

If the stool is of ordinary consistency, it 
should be spread out in a more or less thin 
layer so that the larger particles may be 
easily recognized. If it be, however, a 
watery stool the contents are thoroughly 
mixed and examined as such. 




Fig. 



20. — Boas' stool-sieve. 
(Hemmeter.) 



(2) 



Amount. 

The amount of feces excreted will de- 
pend upon (1) the quantity and quality of the food; (2) the remnants of 
intestinal juices and debris; (3) the condition of the digestive organs, and (4) 
the bacteria. These factors are all important, each being dependent to a 
certain extent upon the other. 

The average moist weight may be considered as varying from 100 to 250 
grams with a dry weight of 20 to 40 grams. This amount may, however, 
reach as high as 20 kilograms as in a case reported by Lynch. 

Number of Stools. 

The number of stools which may be passed in 24 hours is subject to very 
wide variation, even under physiological conditions, but is usually constant 
in the same individual. At least one stool a day should be considered normal, 
although many persons are accustomed to have only one movement of the 
bowels in 48 hours and others in longer periods. We must, therefore, consider 
each individual case before judging as to abnormality in the number of stools. 
Lynch 1 has reported a case of a patient having only one stool each 100 days, 
while Geib and Jones 2 discuss a case in which there was no stool during an 
entire year the patient at the end of that time voiding 32 liters of feces. 

1 Thesis, Buenos Aires, 1896. 

2 Jour. Am. Med. Assn., 1902, XXXVIII, 1304. 



THE FECES. 105 

A diarrhea is said to exist when the stools are frequent and fluid. The 
frequency may vary from 2 to 50 in 24 hours, although a single liquid stool in 
24 hours may constitute a diarrhea in a patient unaccustomed to having a 
movement every day. A normal stool is never fluid, so that the character 
of the stool is of more importance than the number, as individual peculiarities 
must be considered. A diarrhea may be due to increased peristalsis, to in- 
creased intestinal secretion, a diminished gastric secretion, or a decreased 
absorption from the bowel. The most extreme grades of diarrhea are observed 
in Asiatic cholera, dysentery and the summer diarrhea of infants, although a 
marked diarrhea does occur in enteritis, peritonitis, intestinal tuberculosis, and 
uremia. Some infectious diseases are more frequently associated with diarrhea 
than are others, but little of diagnostic importance is obtained from this symptom. 

By constipation is meant the infrequent and irregular movement of the 
bowels, associated with symptoms which are relieved by administration of 
laxatives. The habits of the individual must be taken into consideration 
before judging as to a real or apparent constipation. This condition is physio- 
logically a result of a sedentary life or of a diet lacking in elements which will 
stimulate intestinal peristalsis. 1 Pathologically, we find constipation in cases 
of dilated stomach, occlusion of the bowel, atony of the bowel wall, in con- 
ditions causing increased cerebral pressure, and in obstruction by pressure from 
without the bowel. It must be stated here that we frequently have an apparent 
diarrhea merely as a symptom of constipation high up in the bowel. Such 
being the case, it is better practice to administer a laxative in such conditions 
than it is to give an astringent. 

(3). Consistency and Form. 

The consistency and form of the normal stool vary considerably depending 
upon the nature of the food ingested. The stool is much softer with a purely 
vegetable diet, of which about 85 per cent, is water, than with an animal diet, 
of which only 65 per cent, is water. One differentiates, as regards consistency, 
a well-formed, a mushy, and a fluid stool, between which types there are many 
gradations, some stools being partly formed and partly fluid. Many factors 
may influence the amount of water present in the stool, such as (1) a lessened 
absorption of water from the intestinal canal; (2) intake of a large amount of 
water, and (3) an increased secretion from the intestinal glands. 

The consistency of the normal stool varies from day to day and can be 
constant only when the patient is placed Upon a standard diet. It may be 
abnormally too fluid or too solid, in the latter case being frequently voided in 
the form of very hard masses, known as "scybala." As a general rule, it may 
be stated that the greater the absorption of water from the intestine the more 
firm will the feces be and, in consequence, the more frequently will these scybala 
form. Stools are frequently observed of which the consistency is normal but 
of which the size of the cylinder is quite small. Such small " lead-pencil" 
sized stools have been supposed to be indicative of stricture of the lower bowel, 
but this condition is not necessarily present. 
1 See Cannon, Jour. Am. Med. Assn., 19 12, LIX, 1. 



106 DIAGNOSTIC METHODS. 

Besides the water-content of the feces, the amount of fat, mucus, and 
vegetable residue has much to do with the consistency of the stool. One may 
differentiate the fat from water in the stool by placing a small portion of the 
feces upon a slide and pressing a cover-slip down upon it. If the cover-glass 
remains when the pressure is removed increased fat may be assumed, while 
if the softness of the feces be due to increased water the cover-glass will spring 
away from the feces. 

In some cases, especially those associated with achylia gastrica, the stools 
are very frothy, indicating a marked bacterial decomposition. Such stools 
should not be confused with those of the ordinary diarrhea or with the char- 
acteristic "rice-water" stools of Asiatic cholera, in which particles of mucus are 
readily detected. Such stools are never found associated with large fat contents. 

(4). Odor. 

The peculiar odor of the normal stool is referable to the presence of indol 
and skatol, which arise from the putrefaction of protein material in the large 
intestine. Along with these we may find other substances, such as hydrogen 
sulphid, methane, and phosphine. The odor of the stool is much more 
marked following a meat diet than that after a vegetable diet; it is very slight 
on a milk diet and is practically lost in the fasting condition. If the processes 
in the intestine are of such a nature that fermentation of the carbohydrates 
exceeds the putrefaction of the proteins, the stool shows a distinct sour odor 
traceable to the presence of butyric or acetic acid. The odor in cases of acute 
and chronic diarrhea is frequently very slight, while that of the loose, watery 
discharges of cholera is peculiar and sperm-like, referable to the presence of 
cadaverin. In the diarrhea of children a distinct putrid odor may be present, 
although this is not necessarily the case. The so-called acholic stools have in 
themselves, according to Schmidt, very little odor, showing this property only 
when complicated by decomposition processes arising from the lack of bile. In 
cases of severe dysentery and carcinoma of the large intestine an intensely dis- 
agreeable odor is observed which differentiates these conditions from those 
associated with other types of decomposition. 

(5). Color. 

The color of the stool varies from a light brown to even a black, depending 
upon the kind of food, the residue of the intestinal secretions, the presence of 
pathologic products from the intestinal wall, and the administration of thera- 
peutic agents. The dark color of the normal stool is due to the presence of 
hydrobilirubin, which is formed from the secreted bilirubin by the reducing 
processes in the intestines. Bilirubin is found normally in the stool of a nursing 
child, being converted either into biliverdin or hydrobilirubin under abnormal 
conditions. This change from a light yellow infantile stool to a greenish one 
has much importance to the pediatrician. A well-formed stool is always 
darker in color than is the thin stool, which is equivalent to saying that the 
stool is darker the longer it remains in the intestine. The color of a stool 
under a meat diet is dark brown, with a vegetable diet a lighter brown, and 
following a milk diet a light yellow. This color which is traceable to the diet 



THE FECES. 107 

is shown best in those cases in which bile is excluded from the bowel. The dark 
color of the meat feces is probably traceable to the conversion of the blood- 
coloring matter into hematin and not to the formation of sulphid of iron which 
is so commonly stated. Food products may color the feces a characteristic 
shade. Thus, coffee may give a dark brown color, cocoa a brownish-red color, 
red wine a smoky black-brown color which has a shade of green. Chlorophyll- 
containing plants, such as spinach and lettuce, give rise to greenish shades. 

Occasionally the reduction process in the intestines may go so far as to 
convert the bilirubin into leucohydrobilirubin instead of into hydrobilirubin. 
This hydrobilirubin is identical with the urobilin found in the urine. Such 
stools may be practically colorless when voided, but will be converted into the 
dark brown normal-colored stool on contact with the air. Besides these pig- 
ments, biliprasin has been isolated from the feces by Fleischer, while Miiller 
obtained cholecyanin. 

The variations in color of the stool following administration of therapeutic 
agents is frequently characteristic. Thus, after the use of calomel one ob- 
serves a distinct green coloration due to the conversion of the bilirubin into 
biliverdin. Bismuth preparations color the stool a distinct black, due to the 
formation of the oxid or sulphid of bismuth. Rhubarb, senna, santonin, 
and gamboge cause a distinctly yellow coloration which will change to a 
reddish tone in the presence of alkali. Iron compounds produce shades ranging 
from dark brown to black, while methylene blue gives rise to the production 
of a blue-green color and sandal-wood a reddish-violet color. 
Pathologic Variations in Color. 

Cases showing the presence of large amounts of mucus or of pus in the 
feces are characterized by a gray-white or yellowish-gray coloration of the 
feces. Such cases are seen in membranous colitis and rupture of an abscess, 
especially of the appendiceal variety, into the intestinal tract. In cases of 
syphilitic or carcinomatous ulceration of the colon or rectum this characteristic 
color tone may be more or less influenced by the presence of blood. 

Stools showing the presence of a large amount of fat are clay colored. 
That this coloration is due to the excess of fat, rather than to the absence of 
bile, may be shown by extracting the feces with alcohol and ether, in which 
extraction the bile will be taken up along with the fat and will color the solvents. 
These acholic stools as they have been called occur both in cases associated 
with biliary obstruction and in those showing no obstruction. It would be 
better practice, therefore, to style these stools colorless, instead of acholic 
stools. Strumpell was able to obtain stools of a light brown color by feeding 
patients a diet containing small amounts of fat, thus proving that the increased 
fat was more important than the diminution of bile, although this latter does 
account for some cases. This clay-colored stool may also be found in diarrhea, 
while in Asiatic cholera and dysentery the stools may be absolutely colorless. 
They have also been found in cases of leukemia, carcinoma of the stomach 
or intestine, tuberculous enteritis, and chronic tuberculous peritonitis. The 
cause of this lack of color may be the same unknown cause that produces the 
formation of leucohydrobilirubin spoken of above. 



108 DIAGNOSTIC METHODS. 

In some cases we find a distinct golden-yellow or even a green color of the 
feces. This is due to the presence of unaltered bile, on the one hand, and bili- 
verdin, on the other. Biliverdin is usually found in abnormal decomposition 
processes in the intestine of the infant, while unaltered bile may appear in cases 
of increased intestinal peristalsis, in which the contents of the bowel are rushed 
onward before absorption can take place. This is probably the partial ex- 
planation of the green stool following the administration of calomel. Normally, 
bilirubin is not found in the intestine below the ascending colon. Such being 
the case, it may be possible to judge of the point of irritation in a diarrheal 
attack by the fact that the higher up in the bowel the point of disturbance the 
more of this pigment will be found in the feces. Bilirubin indicates, therefore, 
an enteritis especially of the small, but also of the large intestine. This bile 
pigment may be found most frequently on cellulose material, mucus, muscle 
fibers, and fat. It is readily detected by rubbing up 2 or 3 c.c. of the 
feces with a concentrated aqueous solution of pure mercuric chlorid. This 
mixture is allowed to stand 24 hours and is examined microscopically thereafter. 
The fragments to which the pigment is attached will stain red if due to hydro- 
bilirubin, while those stained with bilirubin will show a green color. Naturally, 
in this examination chlorophyll-containing fragments must be excluded. The 
most favorable material for such examination is the mucus. A green color 
of the stools may also be observed due to infection with the bacillus 
pyocyaneus. 

Blood may give rise either to a distinct red color, a brownish-red shade, 
or even a black tint. If the blood be adherent to the scybalous masses or to the 
well-formed feces, it is usually derived from the rectum or anus and indicates 
hemorrhoids; if it be evenly distributed with the food material and is changed 
from a bright red to a brownish color, it indicates a hemorrhage in the stomach 
or high up in the small intestine, especially if the stool be solid; while an evenly 
mixed bloody feces of fluid character will usually point to the colon as the seat 
of the trouble. As a general rule, it may be said that the darker the color the 
more remote from the rectum will be the hemorrhage. Tarry black blood is 
seldom of low origin, usually indicating trouble in the stomach or duodenum, 
while fluid scarlet blood usually arises from the colon or rectum, although in 
some cases of typhoid fever the blood may be a bright red, although the hemor- 
rhage may be fairly well up in the bowel. In cases of intussusception the blood 
may appear mixed with serum, but with no fecal matter. 

In deciding as to the importance of blood in the feces one must naturally 
exclude that arising from food or from hemorrhages above the gastro-intestinal 
tract. Thus blood coming from the mouth, nose, throat, or lungs may pass 
into the stomach and out with the feces, making a mistake in diagnosis very 
possible. Further, blood arising from vaginal discharges, which may be mixed 
with the feces at the time of defecation, should be excluded. 

The detection of blood in the feces is more or less simple and direct in the 
fresh state, but when intimately mixed with the feces its recognition is a matter 
of some difficulty. In many cases of hidden or occult bleeding blood is never 
detected microscopically or macroscopically. It is practically useless to search 



THE FECES. IO9 

for blood-cells in the feces, as rarely are perfect cells found, unless the blood is 
present in very large amount, many specimens showing no cells. 

Guaiac Test (Van Deen's Test). 

A small portion of the stool is rubbed up with water and one-third of its 
volume of glacial acetic acid added. This mixture is well shaken in a test- 
tube and a few cubic centimeters of ether added. After thoroughly shaking 
this mixture, it is allowed to settle, when the ether, in the presence of blood, 
will have assumed a brownish color. In case the ethereal extract is not clear, 
a few drops of alcohol may be added. On adding to this ethereal extract a 
mixture consisting of equal parts of fresh tincture of guaiac and ozonized 
turpentine, a blue ring will form at the point of contact or a blue coloration will 
be seen throughout the mixture if the tube be shaken. 

This test is much more reliable in its negative phase than in its positive 
phase. The writer has frequently found positive tests for blood arising from 
the employment of tubes previously used with copper solutions or with nitric 
acid, so that he would advise the worker invariably to use either new or thor- 
oughly clean test-tubes when testing for the presence of blood. Moreover, this 
test is given by other substances, which are enumerated in the section on 
Blood and may react positively in the absence of blood. If the patient eats water- 
melon, prunes, potatoes or rice, has been taking iron, or the feces contain much 
pus, a distinct reaction may be present. l In this test as well as in all of the other 
tests outlined, the presence of hematin arising from the meat of the diet must be 
excluded. This can be done only by placing the patient upon an absolutely 
meat-free diet for several days preceding the examination. 

The Schaer-Klunge Test. 

This test is very similar to the preceding, but is much more delicate, being 
positive after the ingestion of only three grams of blood. It is even more 
important when using this test to exclude all hemoglobin and chlorophyll- 
containing foods for some days preceding the examination. The stool is rubbed 
up with water and treated as in the preceding test with acetic acid and ether. 
To this ethereal extract is then added a mixture of 1 c.c. of ozonized turpentine 
and 1/2 c.c. of fresh 3 per cent, alcoholic aloin solution. This may be prepared 
by dissolving what aloin will lie on the point of a spatula in 1/3 of a test-tube of 
95 per cent, alcohol. At the line of contact a distinct red ring will be observed 
in the presence of blood in from three to five minutes. Careful work with this 
test has shown that fat interferes to some extent with its delicacy. It is, there- 
fore, customary to treat the feces with an equal volume of ether and to shake 
thoroughly to remove all the fat present. The ethereal solution is then poured 
off and the remaining fecal material mixed with one-third its volume of glacial 
acetic acid and 10 c.c. of ether, being then thoroughly shaken and set aside. 

A portion of this brownish ethereal extract, which contains the hematin 

formed by the action of the glacial acetic acid upon the hemoglobin of the 

blood, is poured into a thoroughly clean test-tube and treated as above 

1 Boiling the watery suspension of the feces and allowing it to cool before adding the acid 
and ether will prevent reactions arising from ferments and oxygen transmitters other than 
blood. 



IIO DIAGNOSTIC METHODS. 

described with the turpentine and aloin solution. If the tube be shaken after 
the red contact ring has formed the whole mixture will assume a cherry-red 
color. If the tube is allowed to stand for a few minutes the aloin solution may 
sink to the bottom, forming a distinct red layer beneath that of the ether and 
turpentine. A reaction to be positive should appear within 10 minutes as the 
aloin itself will gradually turn red under the conditions of the experiment if left 
for a much longer period. Charcoal instead of carmin should be used to mark the 
feces. The writer has found this test very reliable and very easy of application. 

Weber's Test. 

This test has the advantage of excluding practically every other factor 
which might influence the tests previously given. 1 A portion of the feces is 
extracted with ether to remove the fat and is then separated from the ethereal 
solution. This fat-free feces is then rubbed up with water and treated with 
glacial acetic acid and ether. The ether, as in the preceding test, takes up the 
hematin which is now detected by the spectroscope, showing the characteristic 
spectrum, namely, an intense narrow band in the red between C and D and a 
somewhat more definitely marked group of three broader bands, (i) in the 
yellow, (2) at the boundary between yellow and green, and (3) at the boundary 
between the green and blue, this last band being difficultly recognizable. 

In order to avoid confusion with the spectrum of methemoglobin or of 
chlorophyll, one may convert the hematin into reduced hematin (hemochro- 
mogen) by the addition of alcoholic potassium hydrate, water, and ammonium 
sulphid solution. The spectrum of this latter substance is characterized by the 
two bands in the green. 

This test is probably the most sensitive and should be more frequently 
employed. It is very simple of application, is very reliable and has practically 
no fallacy, especially when the hematin is converted into the hemochromogen. 
This test is, however, not so delicate as the aloin test, so that small amounts of 
blood, as found in occasional cases of occult bleeding, may escape detection. 

Weber originally used the guaiac test with a modification of extracting the 
fat from the feces before testing with guaiac and turpentine. Schumm states 
that if the feces be thoroughly extracted with alcohol and ether most of the fat 
and urobilin will be removed and that under these circumstances Weber's 
test, either with the guaiac tincture and turpentine or with the spectroscope, 
becomes much more valuable. 

Adler's Test. 

O. and R. Adler have introduced the use of benzidin as a test for the 
presence of blood. The stool is extracted with a mixture of alcohol and ether 
for the reasons above mentioned. It is then treated with glacial acetic acid 
and with ether as described in the other tests. This acid ethereal extract, 
which contains the hematin, is then treated with 2 c.c. of a saturated alcoholic 
benzidin solution and 2 c.c. of hydrogen peroxid (3 per cent). In the presence 
of blood a greenish-blue color appears. 

1 Queisser (Therap. Monatsh., 1913, XXVII, 727) found blood by this test in cases fed 
upon file Lenhartz gastric ulcer diet, such cases being free from gastro-intestinal ulceration. 



THE FECES. Ill 

Wagner 1 advocates a so-called "dry test" of the stool which is delicate, re- 
liable and extremely simple. He uses as reagent the Schlesinger-Holst mixture 
of a knife-point of benzidin, 2 c.c. of glacial acetic acid and 20 drops of a 3 per 
cent, solution of hydrogen peroxid. If a few drops of this mixture be poured over 
a little of the solid feces spread on a clean glass slide, a greenish-blue color will 
appear almost immediately in the presence of blood. This benzidin test is 
very sensitive and can be recommended for general use. For a discussion of the 
Phenolphthalin (Meyers' or Boas') test, see Blood. 

(6). Mucus. 

From the diagnostic standpoint the recognition of much mucus in the 
feces is of the greatest importance. Any amount of visible mucus should be 
considered pathological, although it is to be remembered that mucus may be 
increased physiologically as the result of hypersecretion, in which case it will 
appear as a slimy coating of the scybalous masses or as small adherent particles. 
Boas regards the mucus found after strong cathartics as normal, but this is to be 
questioned as the irritation may be sufficient to set up a mild hypersecretion. 
The mucus expelled with the meconium and, according to Lynch, even that 
passed by infants up to the second week of life should be considered normal. 
The fecal mucus is a true mucin, being precipitated by acetic acid, but dissolving 
in 10 per cent. HC1. 

The larger portions of mucus may be easily recognized with the naked eye, 
but the smaller bits are more clearly brought out by rubbing up the feces with 
water and holding the material, in a thin layer, toward the light. If the feces 
be well formed, the mucus may be separated from the exterior of the cylinder, 
as it is never found in the interior of a firm feces. In mushy stools the mucus 
is intimately mixed with the fecal material, usually in the form of smaller 
particles, the exterior type of mucus being of much larger flakes. Nothnagel 
has reported a jelly-like consistency of a mushy stool in a case of jejunal diarrhea, 
in which the mucus was not derived from the intestinal wall, but came, probably 
from the bile. It is important that one be able to recognize mucus, as mistakes 
have been made in the presence of swollen vegetable tissue, fruit pulp, echino- 
coccus membranes, and even of parasites. 

The ordinary form in which mucus appears in the feces is in clumps, 
flocculi, or shreds with irregular margins. These pieces may vary in size from 
those just visible to those several inches in length. In some cases strips, tubes, 
ribbons, or macaroni-like pieces are observed, which are especially frequent in 
enteritis membranacea or mucous colitis. The amount of mucus passed may 
vary from a few flakes to an enormous mass, Bories having seen 120 grams in 
one movement. Occasionally one may see, especially in the stools of infants, 
masses resembling cooked sago granules or "frog spawn," which Kitagawa 
has identified as mucinous material. 

^entralbl. f. Chir., 1914, XLI, 1182; Arch. f. Verdauungskr., 1914, XX, 552. See, also, 
Lyle, Curtman and Marshall, Jour. Biol. Chem., 1914, XIX, 445; Zengerle, Med. Klin., 1914, 
X, 1795; Indemans, Nederl. Tijdschr. v. Geneesk., 1915, LIX, 2077; Roberts, Jour. Am. Med. 
Assn., 1915, LXV, 244; Couturier, Lyon Med., 1914, XLVI, 313, advocates the use of hematin 
as a reagent for occult blood. 



112 DIAGNOSTIC METHODS. 

The consistency of fecal mucus varies from that of a jelly-like mass to 
one having the density of thin leather. The larger the piece the firmer it appears, 
although exceptions do occur. Pure mucus is usually glassy or jelly-like, 
certain inclusions changing its consistency. Cellular inclusions in the mucus 
change it to a paper-like mass, while some specimens appear tenacious due to 
absorption of protein material or to a diminution of its water-content. These 
inclusions, as well as those of fat or bacteria, will diminish the usual transpar- 
ency of the mucus. 

On remaining long in the bowel, mucus may take on the normal brown 
color of the feces due to hydrobilirubin, a dark orange shade from bilirubin, a 
greenish hue from biliverdin, or a red to reddish-brown tinge from blood 
pigments. The usual state of the mucus, however, is colorless. 

It may be stated generally that the most of the macroscopically recognizable 
mucus of the feces arises from the large intestine. Owing to its easy digesti- 
bility the mucus from the mouth, stomach, and upper intestines passes out 
only under conditions of great motility. The secretion of mucus in the small 
bowel is much less than in the large intestine. Of all recognizable forms of 
fecal mucus only the smallest particles arise from the small bowel, and then 
only when they are found in a fluid feces. The higher the secreting point the 
smaller will the particles be, as a rule. These upper intestinal flecks contain 
much detritus of digestion and half-digested cells or free nuclei and crystals 
in cellular form, frequently a few bilirubin granules being observed. 

The mucus from the large intestine, especially from the sigmoid, is large 
in amount, is jelly-like in appearance, and has ordinarily no inclusions. In 
cases of mucous colitis the mucus may be passed in the forms of the large 
strips or bowel-casts, appearing as pure transparent material grayish-white or 
bloody in color and having no inclusions. No fecal matter may be present. 
Such a condition is a pure secretion neurosis and becomes a true inflammation 
only when cell-inclusions are observed. It is to be said that mucus, even with a 
small inclusion of pus or blood, does not necessarily point to ulceration. On 
the other hand, one must not judge from the absence of mucus that no catarrh 
exists, as one frequently finds variations in such excretions. 

Microscopic mucus is much rarer than the macroscopic type, although 
both may be present at the same time. Microscopically, mucus appears as a 
structureless mass, characterized by irregular lines running through it and by a 
difficultly recognizable margin, showing a more or less transparent ground 
substance in which may or may not be found epithelial cells, pus-cells, blood- 
cells, bacteria, protozoa, food remnants, and crystals. If the mucus comes 
from the higher sections of the bowel the food remnants will predominate, while 
in that from the lower bowel the cellular elements are in excess. 

Mucus is stained with difficulty. For a successful stain the reaction must 
be neutral and very little admixture with foreign material is permissible. 
Thionin colors mucin a specific violet, while the other tissue elements are stained 
blue. Methylene blue and methyl violet stain it but slightly, while other aniline 
dyes color only the enclosed cells. Iodin may give a diffuse yellow. 



THE FECES. II3 

(7)- Pus. 

Macroscopically visible masses of pus of a gray-white color are occasionally 
found in the stools, but these may hardly be distinguished from mucus particles 
without the microscope. If in large amounts, attention should be directed to 
the perforation of an abscess into the bowel. Such pus is more or less intimately 
mixed with the fecal material. The passage of pus from the small intestine 
and even from the cecum is associated with such a marked decomposition that 
it can be recognized only with the greatest difficulty. The nuclei may still 
persist, but these resemble very closely those of the food cells. 

A few isolated leucocytes are usually present in the feces, as a result of 
diapedesis through the mucous membrane. A pure pus excretion is never 
seen in cases of uncomplicated catarrh, but in ulcerative processes of the large 
intestine and in many affections of the small bowel (dysentery, ulcerative 
colitis, syphilis, carcinoma, tuberculosis, and typhoid) pus-cells may be found 
in small masses. 

Casein flocculi should be differentiated from pus masses by microscopic 
examination, which will show fat droplets mixed with the albumin. 
(8). Food Remnants. 

The appearance of macroscopic amounts of food remnants in the feces 
is known as lientery and is dependent upon the nature and amount of the food, 
its method of preparation, the degree of its mastication, and upon the condition 
of the digestive organs. 

Much less undigested material remains on a meat diet than after a vegetable 
ration. In the former case we may find small bits of bone, cartilage, tendon, 
hair, feathers, skin, fish scales, and connective tissue; while in the latter we may 
observe cellulose-containing cells, such as those of cereal, cotyledonous and 
leguminous vegetables, skins of fruits, nuts, and vegetables, grape seeds, 
cherry stones, etc. 

Cooking has a great influence upon the digestibility of any food. Boiling 
seems to be the best method, although much of the nutritive material is taken 
up by the cooking water. The method of roasting is such as to produce a 
more nutritive but a less digestible substance, unless it is carried only to the 
point of slight coagulation of the protein. The outside layers of roasted meats 
seem to be practically indigestible, according to the work of van Ledden-Hul- 
sebosch. Smoking seems to be the least desirable form of preparing meats. 
Vegetables become more digestible through the cooking process, owing to the 
bursting of the indigestible cellulose membrane. Those vegetables, such as 
lettuce, cucumbers, onions, turnips, cabbage, and radishes, which are eaten 
raw, appear in the feces absolutely unchanged. 

While great individual differences exist in the power of utilizing digestible 
or undigestible food, yet we must assume that any appreciable residue, especially 
following the Schmidt- Strasburger diet, is pathological. The digestive insuffi- 
ciency may begin in the mouth as a result of too little mastication. The im- 
portance of thorough mastication has been especially emphasized by Fletcher. 
Lack of digestive power of the stomach will have but little influence on intestinal 
digestion, providing the pancreatic secretion is sufficiently active, with the 



114 DIAGNOSTIC METHODS. 

exception that raw or smoked connective tissue will be undigested and will 
appear in the feces. Lientery is much less frequent in motor disturbances of the 
stomach than in those of the intestines. Perhaps the greatest amount of 
undigested residue is seen in cases of perforation of an ulcer or carcinoma of 
the stomach into the intestine, a direct communication being established be- 
tween the stomach and colon. 

Naturally, in cases of increased intestinal motility, one will find more food 
residue in the feces than under normal conditions. This motor insufficiency 
is especially answerable for the appearance of undigested starchy remnants, as, 
even under the most severe conditions, starch digestion is not interfered with to 
any extent in the bowel, providing mastication and preparation of food have 
been sufficient and the succus entericus has not been so diminished by catarrhal 
processes as to permit of fermentation. The direct irritation from the hard 
particles of cellulose may cause an increased peristalsis and, hence, directly 
lead to lientery. 

Marked disturbance of protein and fat digestion, evidenced by an intense 
lientery, will occur if the pancreatic secretion be insufficient. This is even 
more marked if gastric disturbance coexists. A lack of bile will cause an 
exclusive disturbance of fat digestion, which is seldom manifested by the appear- 
ance of macroscopic particles of fat, but is characterized by the typical clay 
color of the stool. 

The final factor which influences lientery is the insufficiency of the 
absorptive power of the intestine. This iucreases the food residue by retarding 
digestion, according to the law that an accumulation of the products of ferment 
activity will prevent further action of the ferment, as well as by holding within 
the bowel the products already digested. 

Only in exceptional cases can one judge of the specific factor at the bottom 
of a lientery, a boundary line between normal and abnormal being drawn with 
difficulty. It is, however, important that one be able to recognize food rem- 
nants both macroscopically and microscopically. The writer recalls a case in 
which the residue of orange and banana pulp was mistaken for a new parasite. 
This recognition of the nature of a fecal residue is especially important in the 
examination of the infantile stool. Normally, the nursing child shows no food 
residue in the feces, but frequently white flecks or clumps are observed, which 
may consist of casein but perhaps more frequently of fat or of soaps. The 
proper recognition of such particles will require both chemical and microscopic 
examination. 

The following discussion of the appearances of the various food residues 
will include both the macroscopical and microscopical examinations, as these 
are inseparable in practical work. 
(A). Protein Residues. 

(a). Muscle Fibers. 

The appearance of muscle fibers in increased amount in the stool is known 

as azotorrhea. 1 The muscle fibers appear as isolated pieces of different size and 

1 Also called creatorrhcea. See Wertheimer, Ztschr. f. klin. Med., 1912, LXXVI, 57; 
Tileston, Arch. Int. Med., 1912, IX, 525; also, Fronzig, Ztschr. f. klin. Med., 1913, LXXVII, 40. 



THE FECES. 



"5 



shape. The smallest pieces have a circular or oval contour, the medium-sized 
particles are jagged, while the larger masses have parallel sides and angular 
surfaces. These fibers are colored yellow or yellowish-brown by the hydro- 
bilirubin, but may be tinged with bilirubin or by foreign pigments. The 
color will depend both upon the amount of pigment and upon the time the 
fibers have lain in the bowel. 

The smaller pieces may be entirely homogeneous, although these as well as 
the larger bits show both transverse and longitudinal striations. If the fibers are 
well digested, the longitudinal striations may be the only ones showing, and even 
these may disappear. No nucleus is visible unless the pancreatic secretion is 
entirely lacking. 1 No specific micro-chemical reactions are known for these 
fibers. They may be tested with any of the color reactions for protein material. 

Connective tissue and elastic tissue fibers are occasionally associated with 
muscle fiber and may be recognized by their appearance. 

(b). Casein. 

This is found especially in the infant stool and is always pathological. 
These masses, known as curds, are more or less round clumps which vary in 
size from that of a pin-head to that of a hazel-nut. They are either pale or 
golden-yellow, the larger masses being a pure white inside. These curds always 
have a distinct yellow tone exteriorly, but the interior is pure white. If these par- 
ticles are pressed between a slide and cover-glass, they spread out like white 
cheese and show absolutely no structure microscopically. These masses 
show the protein reactions. 

Leiner's Test. 

A small amount of fecal matter containing these curds is spread on a slide 
and dried in the air. It is then fixed by heat and stained with a mixture of 
equal parts of a 0.75 per cent, solution of acid fuchsin and methyl green in 50 
per cent, alcohol (dilute mixture 10 times with water). At the end of 15 minutes 
the slides are placed in distilled water and left for one hour. Casein and para- 
casein will take a pale blue or violet color, while similar substances will show a 
greenish tone. 

Microscopically, one may differentiate from these true casein particles 
(curds), certain products which are more or less normal in the stools of the 
child. These masses are smaller than the curds, being not usually over pin- 
head size, are more yellow in color, and appear under the microscope as clumps 
of fatty acid crystals or of fat droplets and bacteria bound together by mucus. 2 

(B). Fat Residues. 

Fatty substances are present in all stools, if there be any in the diet, either 
in the form of neutral fat, free fatty acids, or soap, especially of calcium and 
magnesium. If present in macroscopically recognizable amounts, the con- 
dition is known as steatorrhea. 

Neutral fat may be present in the form of white colorless clumps of different 

1 See Atchley (Arch. Int. Med., 1915, XV, 654), whose experiments tend to disprove the 
value of Schmidt's nuclear test. 

2 See Courtney, Am. Jour. Dis. Child., 1912, III, 1. 



Il6 DIAGNOSTIC METHODS. 

size and irregular in shape, some being globular while others are distinctly 
angular. The more usual form is the refractive, opaque, more or less yellow 
globule. Occasionally the fat may appear as a melted oil which hardens 
over the surface of a formed stool or gives the appearance of vaselin to a semi- 
solid stool. Neutral fat may be recognized by its ready solubility in ether and 
its black color on treatment with osmic acid and deep red color when acted 
upon by Sudan III or Scharlach R. 

Fatty acids appear either as scales or as crystals of varying form. The 
scales may not be distinguishable from those of pure fat except by their easy 
solubility in cold alcohol. The crystals are thin, delicate, curved needles, 
which run to a distinct point and are grouped in thick masses. Other types of 
fatty acid needles may occur, such as the small lancet-shaped plate or those 
closely resembling the soap crystal. These crystals are colorless and are 
soluble in cold alcohol, showing no stain with the above reagents for free fat. 
The fatty acid scales are, however, stained by these dyes. 

The soaps likewise appear in the feces as scales or as crystals. The 
scales are less refractive, more firm, usually more angular, and may be colorless 
or yellow-brown from hydrobilirubin or yellow from bilirubin. The crystals 
appear most frequently as uncolored needles, which are shorter, plumper, not 
so pointed as the fatty acid crystals, and are arranged in clusters. Schmidt 
has described a peculiar form of crystal, which he styles the " cracknel" form, 
a round type with a sunken center and a raised border. These crystals are 
insoluble in ether, do not melt on warming, and are uncolored with stains. 
Treated with acids they form fatty acid crystals. 

Any condition which interferes with the proper absorption of fat by the 
mucosa or lymphatics will lead to steatorrhea. This condition may be observed, 
physiologically, after the ingestion of large amounts of fat, as in the oil treatment 
of gall-stones, but we are little concerned with such findings. It is seen, 
pathologically, in cases of atrophy of the mucosa, in amyloid disease of the 
intestines, tuberculous ulceration, tubercular peritonitis, tabes mesenterica, and 
even in catarrhal enteritis. 

The peculiar, glistening, gray-white, pasty, acholic stool of steatorrhea is 
seen more frequently, however, in cases of biliary obstruction and of pancreatic 
disease. The fat in these cases of biliary stasis is in the usual form, that is 
three-fourths of the ether extract of the bile-free feces is present as fatty acids 
and soaps and one-fourth as neutral fat. In cases of acholia 50 to 80 per cent, 
of the fat will be unabsorbed instead of the normal 5 to 10 per cent. 1 

In the fatty stools of pancreatic disease, the fat is largely present as fatty 
acid, no soap being found. The association of steatorrhea with the absence 
of decomposition products, few bacteria, and the presence of maltose in the 
feces is much more indicative of pancreatic disease than is steatorrhea alone 
(Le Nobel). 

(C). Carbohydrate Residues. 

Starch may be present, in the normal feces, enclosed within plant cells 
which have resisted digestion, but well-preserved starch granules are never 
1 See vonHoesslin and Kashiwado, Deutsch. Arch. f. klin. Med., 1912, CV, 576. 



THE FECES. II7 

found normally. A few partially digested or complete colorless granules may 
indicate no abnormality, but many will point to a hyperacidity or to a disturb- 
ance in the small intestine, especially to an insufficiency of the succus entericus 
leading to the so-called "fermentative dyspepsia" (Schmidt). 

Starch may be detected by the addition of Lugol's iodin solution to a 
small portion of the feces spread upon a slide. A blue coloration will indicate 
starch, while a red tone will show the presence of erythrodextrin. 

The varieties of cellulose-containing substances found in the feces are as 
numerous as are the types of such food material. These have been discussed 
previously and cannot be here elaborated. Certain microchemical tests may 
prove valuable in the identification of such cellulose material. 

Cellulose treated with sulphuric acid and then with iodin solution gives 
a blue color due to the conversion of cellulose into amyloid. 

If a solution of zinc chlorid be allowed to act upon a suspected mass of 
cells, which have been previously treated with Gram's iodin solution, a purple 
coloration will be produced. If a neutral or weakly alkaline solution of Congo- 
red be added to cellulose, it will be stained a distinct red. Cellulose is soluble 
only in an ammoniacal solution of cupric oxid, known as Schweitzer's reagent. 

(9). Biliary Constituents. 

As a rule, the unchanged biliary acids, glycocholic, taurocholic, and 
cholalic acids, are absorbed from the bowel so that they do not appear in 
appreciable amounts in the feces. Schmidt believes that cholalic acid is present 
in slight amounts in all feces and that all the bile acids may be increased in 
pathologic conditions. 

Bile pigments, on the other hand, are always present, chiefly in the form 
of hydrobilirubin, although bilirubin may occur. In pathologic conditions 
we may find, beside these two pigments, biliverdin, bilifuscin, bilicyanin, and 
bilihumin. Stercobilin (urobilin) in the feces is usually an index of blood disin- 
tegration. 1 The tests for these pigments as well as for the free bile acids and 
their salts will be considered in detail in the section on Urine. 2 

In cases of cholelithiasis gall-stones of varying size may be found in the 
feces, although not in every case. It is, therefore, of the greatest importance 
that the feces be carefully examined in suspected cases for the presence of these 
concretions. This is especially the case whenever a severe, colicky, abdominal 
pain of doubtful origin exists. The feces should be well mixed with water 
and passed through a fine sieve which will retain any of the suspected particles. 
A single examination of the feces is not sufficient in such cases unless the stones 
be found. In cases of negative findings, the stool should be searched for at 
least two weeks following a suspected attack. According to Naunyn,only 
the very firm stones will leave the bowel, the softer ones breaking up into small 
bits in the bowel. 

These gall-stones vary from the size of a pin-head to that of a pigeon's egg 
and are found as small crumbling masses or as hard stones which either have a 

1 See Robertson, Arch. Int. Med., 1915, XV, 1072; Ibid., 1915, XVI, 429. 

2 See, however, Jovine, Gazz. d. osp., 19 13, XXXIV, 107 1. For the chemical composition 
of human bile consult Czyhlarz, Fuchs and von Furth, Biochem. Ztschr., 1913, XLIX, 120; 
also, Rosenbloom, Jour. Biol. Chem., 1913, XIV, 241. 



Il8 DIAGNOSTIC METHODS. 

jagged surface or a smooth surface with characteristic facets, indicating the 
presence of many such stones. These stones consist for the most part of 
cholesterin mixed with the biliary pigment, or of compounds of calcium with 
the biliary pigments. These calcareous stones may be combinations of the 
various bile pigments and are always the hard facetted type, while the choles- 
terin stones are softer and may be colorless or tinged with bile pigment. The 
nucleus of these stones is usually a mass of organic detritus, in some cases being 
made up of clumps of bacteria, such as the typhoid bacillus and bacillus coli 
communis. A gall-stone may be usually recognized from its fractured surface, 
but frequently it becomes necessary to submit it to chemical examination for 
purposes of identification. 

In this examination for gall-stones the worker must not be deceived by 
the presence of extraneous substances, such a,s seeds, cherry-stones, fats and 
soaps of high melting point and of masses of impacted feces. In the treatment 
of gall-stones by the use of olive oil many of these soft fatty translucent masses 
appear in the feces which are in no way associated with gall-stones. 

(10). Intestinal Sand and Concretions. 

By intestinal sand we have reference to the small granules or masses of 
inorganic salts which appear in the feces. These masses are very small, may be 
spherical or irregular, are usually hard, usually have a reddish-brown or green 
color and consist of inorganic compounds mixed with organic detritus, especially 
with fat and bacteria. While most of this material is made up of ingested sub- 
stances, we may have in cases of neurasthenia and especially in those associated 
with mucous colitis the excretion of large numbers of these particles as the 
result of a true secretory neurosis. Myer and Cook 1 have recently shown that 
the banana may be a source of this intestinal sand. 2 

The massing together of this intestinal sand may form intestinal concretions 
or enteroliths. As a rule, however, these intestinal concretions have as a 
nucleus some foreign body around which calcium and magnesium salts of 
phosphoric and carbonic acid or ammonium magnesium phosphate have been 
deposited. These enteroliths are usually hard, heavy, and round, being colored, 
as a rule, brownish. 3 A second form of intestinal calculus, known as coprol ths, 
are irregular in shape, usually softer than the enteroliths, and consist of inorganic 
material, mixed with inspissated feces. Neither of these types of intestinal 
concretion have any great pathologic significance, although the possibility must 
be granted that some of them might either form or lodge in the appendix and 
thus be accountable for an acute appendicitis. 

(n). Tissue Fragments. 

The examination of feces for tissue fragments is more or less unsatisfactory, 
owing to the fact that these fragments are difficultly recognizable after being 
partially digested by the juices of the intestinal canal. It not infrequently 
happens, however, especially in carcinoma of the lower bowel, that such frag- 

1 Am. Jour. Med. Sc, 1909, CXXXVII, 383. 

2 Talbot (Jour. Am. Med. Assn., 1913, LXI, 238) shows that the banana is not always the 
source. 

3 See Coerr, Jour. Am. Med. Assn., 1913, LXI, 2238. 



THE FECES. II9 

ments may be obtained and a diagnosis made possible by microscopic exami- 
nation. A diagnosis of malignancy would better not be made unless these 
tissue fragments show typical cellular arrangement, or at least, typical arrange- 
ment of the nuclei. The writer has seen a diagnosis of carcinoma made on the 
basis of a strip of mucus with cell enclosures and would, therefore, warn the 
worker to be on his guard against the possibility of such an absurd mistake. 

III. Microscopic Examination. 

This examination includes the search for many substances which have been 
described under the macroscopic examination, so that these need not be here 
considered. There are, however, some elements, both morphological and 
crystalline, which have not been treated. 

For any success whatever in the microscopic examination of the feces, 
great care must be taken in the selection of the material for examination. This 
is especially true when searching for parasites or their ova, as well as for the 
differentiation between various food particles and secretions from the intestinal 
wall. If the stool be soft and mushy it should be thoroughly mixed by stirring 
and suspicious particles looked for macroscopically. Fresh specimens as well 
as stained preparations are then made and examined both with the low and 
high power. In examining feces for bacteria it is always well to mix the feces 
with water and centrifuge for a few minutes. The bacteria will remain sus- 
pended in this process and the coarser fecal material will be deposited. The 
fluid is then poured off and mixed with equal parts of alcohol, the mixture being 
again centrifuged when the organisms will be found in the sediment. This 
procedure is the one adopted by Strasburger in estimating the proportion of 
bacteria in the dry stool. 

Various microchemical reactions are carried out with isolated fecal 
material and frequently are of great importance. In this procedure a few drops 
of reagent are allowed to flow under a cover-glass which covers the specimen. 
The staining material may be drawn under the cover-glass by placing a piece 
of ordinary filter-paper on the opposite side of the cover-slip. 

If the examination for amebse is to be made it is essential that the stool 
be kept warm, as the characteristic differentiating point of an ameba is its 
active motility. This can be done by placing the feces in a bottle which is 
surrounded by warm water until the examination is made. It is wise, also, 
to make the examination upon a warm stage, although this is not absolutely 
essential unless extended search has to be made for the parasite. There are no 
specific stains for the feces, the ordinary Loffler's methylene blue serving very 
well for a general staining of bacteria, while characteristic stains must be used 
when searching for special organisms. 
Morphological Elements. 

Epithelial cells are present in every specimen of feces. These may be 
squamous in form and are usually found in the mucoid particles of the stool. 
They are especially present in rectal carcinoma and in ulcerative conditions 
of the lower bowel. The cylindrical type of epithelium is much the commonest 
form and is found, also, in association with the mucus. Many well preserve 



120 DIAGNOSTIC METHODS. 

cells may be present if their source is in the lower bowel, but, as a rule, these 
cells show all types of degeneration. They occur in catarrhal inflammations 
of the intestinal mucosa and are rarely associated with pus-cells unless ulceration 
has taken place. If the irritation is in the small bowel these cells are always 
more or less digested and contain bilirubin particles in cellular arrangement, 
while if from the lower bowel the cell is usually intact. 

The presence of red blood-cells is determined more by the chemical ex- 
amination than by the microscopical. It may occasionally happen that these 
cells may be seen, but a good result will depend upon a fortunate selection of the 
material for examination. A few scattered leucocytes are practically always 
found in the feces, 1 and assume pathologic importance only when present in 
large amounts as pus-cells, whose occurrence has been previously discussed. 
Crystals. 

Besides the crystals of free fatty acids and soaps previously mentioned 
we find large numbers of other crystals in the feces. Among these we observe 
neutral phosphates of calcium and of magnesium which appear as wedge- 
shaped crystals occasionally forming rosettes in the former case while in the 
latter the crystals are more in the form of rhombic plates. The ammonium 
magnesium phosphate crystals are practically always present in the feces, 
appearing either as the typical coffin-lid crystal or as irregular fern-like masses. 
These triple phosphate crystals were at one time supposed to be characteristic 
of typhoid stools, but like so many other things their importance has been 
exaggerated. Various other crystals of calcium compounds are observed, such 
as calcium carbonate, calcium sulphate, and calcium oxalate, along with 
calcium salts of unknown fatty acids, which have been described by Nothnagel 
as irregular, oval, or circular masses, either fissured or showing concentric 
striations, and being always bile-stained. The lactate of calcium is seen in the 
form of radiating needles arranged in sheet-like masses in the stools of children 
on a milk diet. 

Cholesterin crystals are found as thin, transparent, rhombic plates with 
notched corners. These crystals do not always appear in the typical shape, 
so that they should be tested by the addition of concentrated sulphuric acid 
when the cholesterin crystals will change from a yellow to a blood-red, violet, 
green, and finally blue color. The Charcot-Leyden crystal appears in typical 
form in the feces as a colorless, diamond, double pyramid-shaped crystal. The 
presence of these crystals is practically characteristic of helminthiasis, although 
they may indicate the presence of any parasite from the least harmful to the 
most pernicious. Hematoidin crystals occur as reddish-yellow rhombic plates 
or as groups of needles or amorphous masses in stools showing the presence of 
blood chemically. These have no especial significance and are not found 
as frequently as are the other blood pigments. 

After the use of bismuth preparations we find the oxid of bismuth appear- 
ing in the form of black irregular rhombic crystals with notched edges. Char- 
coal appears in the form of irregular black masses which are larger and not so 
rhombic in form as are the bismuth crystals. 

1 Albu and Werzberg (Ztschr. f. klin. Med., 1912, LXXTV, 394) call attention to the fre- 
quent presence, in the feces of amebic dysentery, of eosinophile leucocytes and myelocytes. 



THE FECES. 121 

The examination of the feces for the various bacteria as well as the para- 
sites and parasitic ova will be discussed in a special section. Various sub- 
stances appearing in the feces have been mistaken for parasites or their ova 
and these also will be discussed in the later sections. 

IV. Chemical Examination. 

The chemical examination of the feces would naturally embrace both 
the qualitative and quantitative estimation of the products of digestion and 
decomposition as well as the estimation of the undigested portion of the food 
along with the products derived from the gastrointestinal canal itself. Clinic- 
ally, such work is rarely carried out and has questionable value from a diagnostic 
standpoint. However, in following the metabolism in any special case it is 
essential that the absorbed and unabsorbed portion of the food be known. 
This applies more particularly to the proximate food principles, protein, fat, 
and carbohydrate, although in some cases the estimation of the inorganic 
intake and excretion is of first importance. 1 The writer cannot attempt in the 
scope of this work to go into great details regarding the chemical examination 
of the feces, but must limit himself to a few selected topics. 
Reaction. 

The normal reaction of the feces does not vary much from a neutral one, 
although it has a tendency to be slightly alkaline, owing to the presence of the 
alkaline secretions of the intestinal tract. The alkalinity of these secretions 
is diminished both by the combination of the alkali salt with the digestion 
products and by the absorption of some of this alkaline material into the blood. 
This reaction may at times be acid owing to the formation of lactic and butyric 
acids in the fermentative processes, while an increased alkalinity may be 
observed in cases of markedly increased ammoniacal decomposition. A pure 
meat diet gives, as a rule, an alkaline feces, while a pure carbohydrate or fat 
diet will have a tendency to form an acid feces. 

The reaction of the feces under pathologic conditions is of little importance, 
although in typhoid fever the reaction is somewhat more strongly alkaline than 
in almost any other condition. So much depends upon the diet as well as upon 
the condition of the digestive organs that no conclusions at present may be 
drawn from the reaction of the feces. 
Total Solids. 

The normal amount of feces passed in 24 hours ranges from 100 to 250 
grams, of which about 75 per cent, is water and 25 total solids. Although the 
determination of the total solids of the feces has little of importance in itself, 
it is essential that one should know the dry weight of the feces in order that he 
may properly calculate the amount of the various substances in the dry feces. 

In determining the total solids in the feces, the specimen, preferably the 
24-hour specimen or each movement separately, is placed in an evaporating dish, 
covered with a small amount of alcohol, and heated over a water-bath with 
frequent stirring. Small amounts of alcohol should be added as evaporation 

1 See Holt, Courtney and Fales, Am. Jour. Dis. Child., 19 15, IX, 213; von der Heide, 
Biochem. Ztschr., 1914, LXV, 363. 



122 DIAGNOSTIC METHODS. 

proceeds in order to hasten the drying process. It is, moreover, necessary to add 
a small amount (10 to 15 ex.) of dilute sulphuric acid and to mix it thoroughly 
with the feces. This combines with the free ammonia, forming a non-volatile 
salt, and thus prevents loss of nitrogen. If the stool be rich in fats, it is wise 
to add a weighed amount of dry washed sand to make the mass more porous 
and thus permit of quicker drying. When the water has been driven off on 
the water-bath, the specimen may be placed in the drying oven at 105 and left 
for several hours, after which it is placed in the desiccator and dried to constant 
weight. Knowing the weight of original substance taken and the weight of the 
dry substance, it is very easy to determine the percentage of total solids. 

An increased total solids of the feces is observed in most cases of con- 
stipation, while in diarrhea the solid residue is much diminished. Increased 
separation of fluid into the bowel and diminished absorption from the bowel 
are the factors regulating the fluid content. Nothing can be determined, how- 
ever, by the examination of the amount of total solids, as regards the pathologic 
conditions accountable for an increase or a decrease. 

Total Nitrogen. 

The determination of the total nitrogen must always be made in estimating 
the metabolism in any given case. In so doing it is likewise essential that one 
should know the absolute nitrogen intake, as without such a factor nothing can 
be learned from the excretion either in the urine or in the feces. The daily 
excretion of nitrogen in a fasting condition varies from 1 to 4 grams, on a mixed 
diet this may run from 3 to 7 grams, while on a vegetable diet the nitrogen 
of the feces may be as high as 10 grams, a pure meat diet yielding between 2 
and 6 grams. In pathologic conditions this amount is practically always in- 
creased, due both to lack of digestive power and to decrease in the absorptive 
function of the intestines. Outside of metabolic experiments the absolute 
amount of nitrogen of the feces has no great diagnostic importance beyond show- 
ing some perversion of intestinal activity. 1 In insufficiency of the pancreatic 
secretion, we are apt to find the greatest loss of nitrogen by way of the feces. 

The method for determination of total nitrogen will be given in detail in 
the section on Urine to which the reader is referred. 

The constituents which go to make up this total nitrogen are the various 
undigested protein bodies, such as albumin, globulin, nucleoprotein, nucleo- 
albumin, and gelatin, along with partially or completely digested products 
of protein origin, bacteria, and secretions from the intestines. These latter 
bodies embrace albumoses, peptone, the various amino-acids, and the hexone 
bases along with a certain amount of ammonium salts. The chemical properties 
of these substances, as well as the methods of their chemical detection and 
estimation, must be looked for in works on physiological chemistry. The 
bacteria form a large percentage of the total nitrogen. 2 It is rare, even in 
metabolic work, that the nitrogen partition of the feces is determined. Much 

1 See McCrudden and Fales, Jour. Exper. Med., 1912, XVII, 20 and 24; also Koplik and 
Crohn, Am. Jour. Dis. Children, 1913, V, 36; Gamble, Ibid., 1915, IX, 519; Van Slyke, 
Courtney and Fales, Ibid., 533. 

2 Mattill and Hawk (Jour. Exper. Med., 191 1, XIV, 433) show that the bacterial nitrogen 
constitutes 53.9 per cent, of the total fecal nitrogen. 



THE FECES. 123 

more importance attaches, with our present knowledge, to such division of the 
nitrogenous material of the urine. Likewise the products of abnormal de- 
composition of protein material, taking place in the bowel with the formation 
of such products as indol, skatol, and phenol, are rarely searched for in the feces. 1 
Our clinical knowledge of the importance of these substances is confined largely 
to their detection and estimation in the urine. In the study of the metabolism 
of various cases of cystinuria, which is associated with abnormal protein disin- 
tegration, certain diamines, such as cadaverin and putrescin, have been isolated 
from the feces by Udransky and Baumann. 2 
Fat. 

The chemical estimation of the amount of fat is of importance only in 
metabolic work, as in the more direct clinical examinations this substance is 
detected by macroscopic and microscopic methods. It is, however, essential 
that the loss of fat by way of the feces should be known before a proper metabolic 
balance can be struck. 

The dried feces is treated with a small amount of 1 per cent, acid- 
alcohol and evaporated to dryness in order to convert any soaps which may be 
present into the fatty acids. The dry residue thus treated is then placed in a 
Soxhlet apparatus and extracted with ether for at least 72 hours. The ether, 
which has taken up the free fat and the fatty acids, is then evaporated and the 
residue of fats weighed. From this weight the percentage of fat may be deter- 
mined, and, knowing the original dry weight of the total 24-hour feces, the 
total fat lost by way of the feces may be readily calculated. 3 

The amount of fat in the feces will depend much upon the amount and 
quality of the diet. In fasting conditions the amount is usually about 1 gram. 
On diets poor in fat the amount in the feces may exceed that of the diet, thus 
indicating a loss of fat from the body. It seems to be a general rule that the 
higher the melting point of the fat of the diet the greater will be the loss in the 
feces Thus on a diet containing only butter as a fat about 4 per cent, of the 
intake will be ost, while with pork fat the loss may be as high as 13 per cent. 
It is impossible to say, a priori, just what the fat content of a normal feces would 
be. With the ordinary diet of our country it would range from 2 to 7 per cent, 
of the intake, which should be somewhere about 100 grams of fat per day, 
representing a loss of from 2 to 7 grams daily. 
Carbohydrates. 

As previously stated, carbohydrates may appear in the feces either in the 
form of starch or of cellulose. Only in exceptional conditions do we find any 
of the monosaccharids present. Disaccharids, such as lactose and maltose, 
may be occasionally found, but only when there is a combination of insufficiency, 
both of a salivary and pancreatic ferment. The presence of any appreciable 

1 See Moewes, Ztschr. f. exp. Path, und Ther., 1912, XI, 555. See, also, Rodella, Arch. f. 
Verdauungskr., 1014, XX, 657; Whipple (Jour. Am. Med. Assn., 1915, LXV, 476) believes 
that the intoxication of intestinal obstruction is due to the absorption of a primary proteose. 

2 See Hopkins (Arch. Int. Med., 1913, XI, 300) for a discussion of the presence of hemo- 
lysins in fecal extracts. 

3 See Cowie and Hubbard, Am. Jour. Dis. Child., 1913, VI, 192; also, Bloor, Jour. Biol. 
Chem., 1913, XV, 105; Ibid., 1913, XVI, 517; Saxon, Ibid., 1914, XVII, 99; Gephart and 
Csonka, Ibid., 1914, XIX, 521. 



124 



DIAGNOSTIC METHODS. 



amount of starch granules must be considered pathological, while the amount 
of cellulose will depend upon the amount in the food as well as upon the 
preparation of the food and its mastication. 

Nothing is to be learned, from the clinical standpoint, by the determination 
of the absolute amount of carbohydrate in the feces. In the study of the 
utilization of food substances by the system and in general metabolic work 
it is, however, necessary to know just how much of the carbohydrate intake is 
absorbed. This may be determined indirectly by subtracting from the weight 
of the dry feces the sum of the protein, fat, and ash. This result will represent a 
much higher figure for carbohydrates than the one obtained by direct deter- 
mination. In the direct determination much difference exists between the 
soluble forms and the insoluble cellulose. In the latter case we have to do 
with a useless form of carbohydrate and should, therefore, direct our attention 
rather toward the estimation of the amount of undigested or partially digested 
starch. We should determine the amount of cellulose and subtract this factor 
from the total carbohydrate obtained in the previous subtraction. This cor- 
rected figure will represent more nearly the true amount of 
carbohydrate than will the former, although for scientific 
purposes a direct determination is essential. 

The starch may be estimated by taking a weighed 
amount of the dry feces and treating with 50 c.c. of 10 per 
cent. HC1. This is then boiled for one-half hour in order to 
convert the starch and digested portions of starch into the 
monosaccharids. This acid solution is then filtered and 
washed with sufficient water to make the total approximately 
the same as that of the original solution. The filtrate is then 
neutralized with sodium hydrate and made up to exactly 100 
c.c. in a volumetric flask. The sugar in this solution is then 
determined by the methods outlined under Urine. This 
method, although not absolutely accurate will yield results 
sufficiently correct for ordinary purposes. More accurate 
results will be given by the Volhard-Pfl tiger method to be 
discussed later. The amount of glucose, as determined by 
either of these methods, will give, if multiplied by 0.94, the 
amount of starch in the feces taken. The percentage may 
then be readily calculated and the total quantity of the 24-hour 
excretion of starch determined. 

The fermentation method of Schmidt was advanced to 
permit of rough estimation of the presence of pathological 
amounts of starch, which, under ordinary circumstances, 
should have been digested. The principle of the method 
is the estimation of the amount of gas produced by the action of the 
intestinal bacteria upon the sugar which is formed from the starch by the 
intestinal amylolytic ferments. Prior to this determination the patient is 
placed upon the test diet outlined on page 100. The test is made as follows: 
Approximately 5 grams of the moist stool are placed in the vessel a which is then 



Fig. 21. 
Schmidt's fermen 
tation apparatus. 



THE FECES. 125 

filled with water and the contents thoroughly mixed (see cut) . The stopper is 
then placed in the fermentation flask in such a way that no air-bubbles are left. 
The tube b is filled with tap-water and closed with a small stopper without 
the inclusion of any air-bubbles. The tube c is now placed in position as shown 
in cut and the whole apparatus put in an incubator for 24 hours. The tube c 
has a small opening in the top so that water may be readily forced from the tube b 
into c by pressure of the gas produced. According to Schmidt 0.1 gram of starch 
will cause the tube b to show about one-half its volume of gas. Normally, a 
positive result is said to occur when the tube is one-fourth to one-third filled 
with gas. The material in the fermentation flask a should be tested with 
litmus-paper after the test is complete to obtain the reaction of the mixture. 
If the gas formation is, as it should be, due to carbohydrate fermentation 
there will be a slight increase in the acidity of the mixture, while if it be due 
to protein putrefaction the reaction will show a slightly increased alkalinity. 

The various decomposition products of carbohydrate digestion and 
fermentation are rarely of importance in fecal examinations. These products 
consist of volatile fatty acids, lactic acid, saccharic acid, alcohol and aldehyde, 
and the conjugated glycuronic acids. Tests for these various substances will 
be found in various parts of this work or in works on physiological chemistry 
to which the reader is referred. 1 

From the clinical standpoint the gases produced by the processes of 
fermentation and putrefaction in the bowel have little value. The same is to 
be said of the quantitative estimation of the inorganic constituents of the feces. 
These latter may be determined by methods outlined in quantitative analysis. 

The various ferments found in the intestinal canal may be detected in the 
feces, but such examinations are at present of little clinical value. Methods of 
isolating these ferments and of determining their activity belong more to 
physiological chemistry than to clinical diagnosis and will, therefore, be 
neglected. 2 

Phenoltetrachlorphthalein Test. 

There is no question but that the liver has a more varied and manifold 
functional activity than any other organ of the body. Slight disturbances in 
hepatic activity are a matter of relatively frequent occurrence, but these are not 
attended with any marked perversion of function as the larger portion of the 
parenchyma of the liver may not be involved. In disease, however, of the liver 
the character of the functional changes will vary with the nature of the under- 
lying pathological condition. There is not so much difficulty in determining, 
from clinical and laboratory data, that hepatic disease exists as there is in estimat- 
ing the intensity of the disease and the degree of disturbance of the functional 
activity. For this reason various attempts have been made to devise tests 
which would indicate such functional variations sharply and permit of a judg- 
ment concerning their intensity. While many of these tests have been proven 

1 See Fischer, Ztschr. f. exper. Path. u. Therap., 1913, XIV, 179. 

2 See Crohn, Am. Jour. Med. Sc, 1913, CXLV, 393; also, Chace and Myers, Arch. Int. 
Med., 1913, XII, 628; Brown, Bull. Johns Hopkins Hosp., 1914, XXV, 200; Einhorn, Am. Jour. 
Med. Sc, 1914, CXLVIII, 490; Crohn, Ibid., 839; Arch. Int. Med., 1915, XV, 581. 



126 DIAGNOSTIC METHODS. 

of questionable value, some of them, such as the levulose and galactose tolerance 
test as well as the urobilinogen estimation, are of some importance in diagnosis 
and prognosis and will be discussed in the section on Urine. These tests have, 
for the most part, lacked a quantitative element, indicating more the presence 
than the extent of the disease. 

Rowntree, Hurwitz and Bloomjield have introduced a test for the functional 
activity of the liver, which is probably the best at our disposal. They take 
advantage of the fact, discovered by Abel and Rowntree, that phenoltetrachlor- 
phthalein, a drug first prepared by Orndoff and Black, escapes from the body 
only in the bile and may be recovered from the feces to a considerable extent. 
Under normal conditions the drug is not excreted to any appreciable extent in 
the urine. In other words, the liver exerts a specific action in excreting this 
dye, just as does the kidney in eliminating phenolsulphonephthalein. With this 
fact as a basis, these workers were able to show that a decreased capacity for 
its excretion was a resultant of lowered functional activity due to disease, and 
that minimal amounts of the dye escaped with the urine in pathological condi- 
tions of the liver. By direct estimation of the amount of this dye excreted 
by the bile and carried into the intestines one may readily estimate the presence 
and, to some extent, the degree of the functional disturbance of the liver. 

The solution used for the injection is an aqueous solution of the disodium 
salt of phenoltetrachlorphthalein. This is prepared as follows: 2.5 grams of the 
phenoltetrachlorphthalein are placed in a 200 c.c. flask with 5 c.c. of an 8 per 
cent. (2N) sodium hydrate solution and 45 c.c. of freshly distilled water. 
Boil for 20 minutes under a reflux condenser. Filter the solution into a 100 c.c. 
flask, when it is ready for use. It is an intense purplish-red solution, which 
does not keep for many days owing to the precipitation of the dye by the C0 2 
of the air. Hence relatively fresh solutions must be used. 

Method of Administration. 

Eight c.c. of the above solution, representing 400 mg. of phenoltetrachlor- 
phthalein, are administered intravenously to the patient, using all the antiseptic 
and aseptic precautions which this method involves. The funnel and tubes 
are filled with sterile physiologic salt solution and, after the flow is established, 
the solution of the dye is added. Fifty to 100 c.c. of salt solution are used, the 
dye being washed in with the salt solution until the fluid entering the vein is 
colorless 

Collection of Material. 

Before beginning the administration of the dye, active purgation of the 
patient should be induced and should be continued throughout the time of 
observation. If little feces can be obtained, enemata should be employed. 
Collect all the fecal material which passes for 48 hours and the urine for 24 
hours. 

Place the total 48-hour specimen of feces in a large bottle and dilute it with 
1 to 1.5 liters of water, depending on the amount of feces. Place this mixture 
in a shaking machine for 5 to 20 minutes. Without allowing time for sedimen- 



THE FECES. 127 

tation, one-tenth of the total mixture is placed in a liter flask and to this is added 
5 c.c. of 40 per cent, sodium hydrate, which causes the mixture to take on a 
dirty red color. Now dilute the mixture to 1 liter with water, stopper the flask 
and shake thoroughly. One hundred c.c. of this preparation are placed in a 
200 c.c. flask and 5 c.c. of a saturated solution of basic lead acetate are added. 
Now add 5 c.c. of 40 per cent, sodium hydrate solution to elicit the maximum 
intensity of red color. If the amount of NaOH added does not seem suffi- 
cient, add a little more, being careful not to redissolve the precipitate present. * 
Make up the contents of the flask to 200 c.c, shake and filter a small portion 
for comparison with a standard solution of phenoltetrachlorphthalein. This 
comparison is made in a colorimeter, either of the Duboscq type or of the type 
of Rowntree and Geraghty's modification of the colorimeter of Autenrieth- 
Konigsberger. For comparison one may use 0.4 c.c. of the original solution, 
making this up to a liter and adding enough NaOH to insure the maximum 
color. 

There are certain elements connected with this test that are not very desirable. 
It involves intravenous injection, which is followed in many cases by thrombosis 
at the site of injection. This is not sufficient to occasion serious discomfort, 
but the fact should be remembered and its possibilities taken into consideration. 
Further, it necessitates a careful collection of feces for 48 hours. The quality 
of the red color obtained in the fecal extract in some cases is such that accurate 
quantitative determinations are difficult. 

The results with this test show that a phenoltetrachlorphthalein output in 
the feces below 30 per cent, or its appearance in the urine is quite infrequent in 
health. The output of a less amount is relatively frequent in hepatic disease, 
although a normal output of 30 per cent, does not exclude liver lesions. The 
output of the dye in the feces is independent of the quantity of bile excreted, 
although it is evident that none of the dye will reach the intestines in cases of 
obstruction of the duct. Functional changes, as evidenced by a lessened output 
of the dye in the feces, have been most marked in cirrhosis, carcinoma and in 
cachexias associated with severe anemias. Chronic passive congestion does not 
give rise to a much changed excretion, although marked hepatic congestion 
associated with insufficiency of the myocardium is usually accompanied by a 
lowered output. Although this test is not to be regarded as diagnostic in all 
cases of perversion of hepatic activity, yet it offers much evidence which can be 
obtained by very few other tests so far advanced. 2 

1 If difficulty is experienced on account of the quality of the color at this point, add to the 
100 c.c. of mixture 5 to 10 c.c. or more of a calcium chlorid mixture (CaC^, 90 grams; cone. 
NH4OH, 10 c.c; water, 50 c.c.) until the best color appears. Dilute to 200 c.c, allow the 
mixture to stand from 1 to 24 houis and decant the supernatant fluid for comparison. 

2 Orndoff and Black, Jour. Am. Chem. Soc, 1901, XLI, 349; Abel and Rowntree, Jour. 
Pharmacol, and Exper. Therap., 1909, I, 233; Whipple, Mason and Peightal, Bull. Johns 
Hopkins Hosp., 1913, XXIV, 207; Rowntree, Hurwitz and Bloomfield, Ibid., 327; Whipple, 
Peightal and Clark, Ibid., 343; Whipple and Christman, Jour. Exper. Med., 1914, XX, 297; 
Chesney, Marshall and Rowntree, Jour. Am. Med. Assn., 1914, LXIII, 1533; Sisson, Arch. 
Int. Med., 1914, XIV, 804; Whipple and Speed, Jour. Exper. Med., 1915, XXI, 203; McLester 
and Frazier, Jour. Am. Med. Assn., 1915, LXV, 383; Kahn and Johnston, New York Med. 
Jour., 1915, CII, 848. 



128 DIAGNOSTIC METHODS. 



V. Bacteriology of the Feces, 



The bacteria of the intestinal canal are of many types, many of which are 
purely saprophytic, while others may or may not be pathogenic. 1 The number 
of these bacteria is usually enormous. One may determine the amount of these 
bacteria by the method of Strasburger, who uses the following technic: Two 
c.c. of feces are rubbed up in a porcelain mortar with 30 c.c. of 1/2 per cent, 
hydrochloric acid. This mixture is then placed in centrifugal tubes and whirled 
for one minute. The bacteria will remain in suspension in the liquid which is 
poured off from the sediment. The sediment is again rubbed up with a little 
hydrochloric acid and again centrifuged, the liquid being added to the first 
portion. This procedure may be repeated until the fluid no longer becomes 
turbid on centrifuging. This acid solution, holding in suspension the bacteria, 
is then mixed with an equal portion of ordinary alcohol and placed in a beaker 
which is allowed to remain on a constant water-bath at 40 for 24 hours. At the 
end of this period a portion of the fluid may be evaporated and more alcohol 
added. This mixture is then placed in the centrifuge tubes and whirled for 
several minutes. The bacteria are now deposited and the supernatant fluid is 
poured off and mixed so that it may be again centrifuged. The bacterial sedi- 
ment is washed with alcohol and ether in the centrifuge tube and is then placed 
in a weighed dish. This is dried at ioo° and again weighed. In order to de- 
termine the amount of bacteria as compared with the total feces, the dry weight 
of 2 grams of the fresh feces is determined as previously outlined. Knowing 
these factors, the percentage of bacteria in the dry feces may be easily calculated. 
Strasburger found that this was approximately one-third the weight of the dry 
stool and represented about 8 grams per day. 2 As this dry feces is used for the 
determination of total nitrogen, we must bear in mind that the bacteria of the 
feces will represent about one-half of the total nitrogen of the feces. This fact is 
especially important in metabolic work. 

It is to be said that the bacterial flora of the intestine is so varied that very 
careful and long-continued work is necessary to isolate a special organism. 
From the standpoint of preventive medicine it should always be remembered 
that the feces contain large numbers of typhoid bacilli and cholera spirilla, so 
that measures should be taken to treat properly such ejecta as soon as voided. 
While the majority of the bacteria of the intestines are saprophytic, being intro- 
duced with the food or drinking-water, yet we find theBacilluscoli communis as 
a normal habitant of the intestine. This organism is usually harmless, but may, 
under certain conditions, become distinctly pathogenic, many obscure cases of 
gall-bladder infection, for instance, being traceable to this organism. 

The presence of non-pathogenic organisms seems to be an essential for 
proper performance of intestinal function. This idea, originally advanced by 
Pasteur, has been denied by Schottelius, Nuttall, and Thierfelder, although 
their experiments extended only over periods of 17 days. These intestinal 

1 See MacNeal and Chace, Arch. Int. Med., 1913, XII, 178; also, MacNeal, Am. Jour. 
Med. Sc, 1913, CXLV, 801; Rahe, Jour. Infect. Dis., 1915, XVI, 210. 

2 See Mattill and Hawk, Jour. Exper. Med., 191 1, XIV, 433. 



THE FECES. 129 

bacteria not only aid digestion, but also prevent a certain amount of abnormal 
decomposition, owing to the fact that they inhibit the development of foreign 
types to a large extent, especially under normal conditions. These normal 
bacteria may under certain conditions, however, become pathogenic, but only 
when the intestinal wall loses its continuity. In case the normal bacteria of 
the intestine become too numerous, the products formed by their activity upon 
protein material may be absorbed and bring about certain toxic effects. 1 It is 
interesting in this connection to find that the bacteria are diminished in amount 
in chronic constipation, according to Strasburger. The toxic symptoms must, 
therefore, be referable to the absorption of other than bacterial products of 
decomposition, or the products have increased toxicity. For the best conditions 
to exist the symbiotic relations of the intestinal bacteria should be such that 
there are neither too few nor too many bacteria. 




Fig. 22. — Cholera spirilla. {Pit field.) 

The normal adult stool shows a preponderance of Gram-negative organisms 
(colon bacilli). Pathologically an increase in this type may be noted (cholera 
spirilla) or Gram-positive organisms may prevail. This Gram-staining varia- 
tion in the bacteria of the feces is, also, largely influenced by changes in diet, 
bacteria of the Bacillus acidophilus and bifudus types becoming quite prominent 
under a substitution of a low protein for a high protein diet. 2 

(a). The Cholera Spirillum. 

This organism, known as the comma bacillus, is about 2 microns long 
and 1/2 micron thick. It is very actively motile and has a single delicate 
flagellum at one end. It stains easily with the ordinary bacterial stains and is 
decolorized by Gram's method. The cultural peculiarities of this spirillum 
will be discussed in the last section of this book. This organism is usually 
recognizable in the stools of Asiatic cholera, which are the characteristic "rice- 
water" stools. The blood serum of patients affected with, cholera will show 
very characteristic agglutination of cultures of these organisms. The blood 

*See Zuntz, Die Naturwissenschaften, 1913, I, 7. 

2 See Herter and Kendall, Jour. Biol. Chem., 1910, VII, 203; Rettger and Horton, Centralbl. 
f. BakterioL, Orig., 1914, LXXIII, 362; Osborne and Mendel, Jour. Biol. Chem., 1914, 
XVIII, 177; Logan, Jour. Path, and Bacterid., 1914, XVIII, 527; Rettger, Jour. Exper. Med., 
1915, XXI, 365; Torrey, Jour. Infect. Dis., 1915. XVI. 72. 



13° 



DIAGNOSTIC METHODS. 



is usually used in a dilution of i to 15, the reaction being observed in from 5 to 
20 minutes. 

Closely related to this comma bacillus of Koch is the bacillus of Finkler- 
Prior. This latter organism may be distinguished from the spirillum of Asiatic 
cholera by its morphology, the organism being larger and thicker than the 
comma bacillus, and by the appearance of the stab cultures on gelatin. The 
cholera spirillum forms a typical funnel-shaped depression, while the bacillus 
of Finkler-Prior shows a stocking-like depression. The Finkler-Prior bacillus 
is found in cases of cholera nostras. It may be necessary for the absolute 
identification of these organisms to apply bacteriolytic tests with serum of 
animals immunized against a specific type of organism. 1 (See Chapter XI.) 




m : m 



Fig. 23.- 



-Bacillus typhosus, stained to show flagella. 
(Oertel after Frankel and P/eiffer.) 



(b). Typhoid Bacillus. 

This organism, discovered by Eberth, is so similar in morphology to 
numerous other organisms, especially to the Bacillus coli communis, that simple 
staining methods do not suffice for its detection. The characteristic stool of 
typhoid fever is a copious watery stool, having a strong odor and an alkaline 
reaction. This stool is known as the "pea-soup" stool, and may be tinged with 
blood and contain many pus-cells. « 

The typhoid bacilli are medium-sized organisms with rounded ends, 
generally short, but sometimes long or thread-like and frequently showing 
faintly stained, sharply defined areas in their protoplasm. They are actively 
motile and have both polar and lateral flagella. This organism stains with the 
ordinary dyes and is decolorized by Gram's method. 

Method of Drigalski and Conradi. 2 

Three pounds of minced beef are mixed with 2 liters of water and allowed 

1 See Public Health Reports, 1912, XXVII, 371. 

2 Ztschr. f. Hyg. u. Infektionskrankh., 1902, XXXIX, 283. For a discussion of the use of 
benzin in differentiating members of this group see Benians, Ztschr. f. Chemotherap., 19 13, II, 
28; Bierast, Centralbl. f. Bakteriol., I. Abt., Orig., 1914, LXXIV, 348; Schmitz, Munch, 
med. Wchnschr., 1914, LXI, 2115; Jaffe, Wien. klin. Wchnschr., 1915, XXVIII, 418. 



THE FECES. I3I 

to stand overnight. The beef is then pressed and the juice boiled for one 
hour and filtered. To the filtrate are added 20 grams of Witte's peptone, 
20 grams of nutrose, and 10 grams of sodium chlorid. Boil this mixture one 
hour and filter. To the filtrate 60 grams of agar are added and the mixture 
boiled for three hours, one of which should be in the autoclave. Slightly 
alkalinize the mixture to litmus-paper, filter, and boil for one-half hour. To 
this hot agar solution, which should be now about 6o°, add the following litmus- 
lactose solution. Two hundred and sixty c.c. of litmus solution are boiled for 
10 minutes, after which 30 grams of chemically pure lactose are added and 
the mixture boiled for 15 minutes longer. This litmus-lactose solution is 
added while boiling to the hot agar solution, the mixture being well shaken and 
again faintly alkalinized to litmus. Four c.c. of a hot sterile 10 per cent, solution 
of sodium carbonate and 20 c.c. of a freshly prepared 0.1 per cent, solution 
of crystal-violet B. (Hochst) in warm sterile distilled water are then mixed 
in. This medium may be poured directly into plates or kept in flasks. It 
soon hardens to a firm mass and does not become dry readily. 

The principle upon which the use of this medium depends is that in 
the presence of both lactose and protein the colon bacillus will first attack 
the milk-sugar, while the typhoid bacillus will act upon the protein. In the 
presence of litmus the colonies of colon bacilli become distinctly red, while 
those of the typhoid bacillus are blue. The crystal-violet inhibits the growth 
of many of the other organisms, especially of the acid-producing type. 

If the stool be fluid, as is usually the case, one series of two plates is inocu- 
lated with the undiluted stool, another with a stool diluted with 10 volumes 
of sterile normal salt solution, while other dilutions, such as 1 to 100 and 1 to 
1000 may also be made. If the stool is solid it is rubbed up into a homogene- 
ous mass with sterile salt solution and the various dilutions made as above. 
In making the inoculations from the stools, the material is rubbed over the 
surface of the medium, the plates being left open to allow the surface to dry. 
The dry plates are then placed in the incubator at 37 and examined at the end 
of 24 hours. Any contamination of the media will be killed by the crystal- violet. 

It frequently happens that certain strains of the paratyphoid bacillus 
develop blue colonies in this medium. It is, therefore, necessary for absolute 
differentiation that the agglutination test described under blood be carried out. 
In this way the typhoid bacillus may be absolutely identified, especially in 
the presence of its well-known morphological characteristics. 
Method of Kendall and Day. 

This method 1 permits of rapid diagnosis and is especially of importance 
in the examination of stools of suspected "carriers." The culture medium is 
a modification of that of Endo. Plain nutrient sugar-free agar is prepared as 
follows: 1000 c.c. of cold tap water, 15 grams of powdered agar, 10 grams of 
Witte's peptone, and 3 grams of Liebig's extract of beef are mixed and cooked 
in the double boiler for one hour. Just alkalinize to litmus by careful addition 
of sodium hydrate solution, cook for 15 minutes and filter through absorbent 

x Jour. Med. Research, 1911, XXV, 95. See, also, Schmidt, Deutsch. med. Wchnschr., 
1915, XLI, 33; Hirschbruch and Diehl, Ibid., 606. 



132 DIAGNOSTIC METHODS. 

cotton. This nitrate is then stored in flasks containing known amounts (100 
c.c.) and is sterilized in the autoclave. When it is desired to use this media, 
add 1 gram of chemically pure lactose to each 100 c.c. and heat in the ster- 
ilizer until the media is melted and the lactose thoroughly distributed. Now 
prepare a mixture of 1 c.c. of a filtered 10 per cent, alcoholic solution of fuchsin 
and 10 c.c. of a fresh 10 per cent, aqueous solution of sodium sulphite. Add 
1 c.c. of this sterilized and decolorized fuchsin mixture to each 100 c.c. of the 
liquid lactose media and mix thoroughly. Plates are poured and allowed to 
harden (with the covers removed) in the incubator for 30 minutes, when they 
are ready for inoculation. 

A small portion of feces is emulsified in 10 c.c. of sugar-free 1 bouillon 
and incubated for 1 hour at 37 C. This suspension is rubbed gently but firmly 
over the surface of the agar plates by means of sterile hooked glass rods and in- 
cubated for 18 hours at 37 C. The translucent, colorless, "dewdrop" colonies 
are removed to small tubes, which contain 1 c.c. of sugar-free bouillon and have 
been kept at 37 C. prior to use. 2 Incubate for 2 hours and make agglutina- 
tion tests, using a known serum of high agglutinating power (see blood). 

(c). The Bacillus of Dysentery. 

Bacillary dysentery is distinctly different from the amebic type of dysentery. 
The Bacillus dysenteriae, or Shiga's bacillus, is now generally recognized as 
the specific organism of this type of dysentery. 

Flexner found a similar bacillus in the dysentery of the Philippines, while 
Kruse has found practically the same type in Germany. In the United States 
Flexner and Harris find an organism which answers the description of the ordi- 
nary Shiga bacillus. The variation between the different organisms described 
is very slight, so that one may generally consider them as varieties of the same 
species, although certain cultural and agglutinating differences are observed. 

The Shiga bacillus is a short rod with rounded ends, much resembling the 
typhoid bacillus but much plumper. It does not seem to have very active 
motility as far as progression is concerned, although it does show a high degree 
of molecular motility. It stains with the usual dyes and is decolorized by 
Gram's method. The only sure method of identification of the various types 
of the dysentery bacillus seems to be the agglutination test. 

The fecal material is best obtained by curettage of the rectum, as these 
bacilli seem to be present in the mucus and are thus more easily concentrated. 
The cultural peculiarities of this organism will be found in a later section. 

(d). The Tubercle Bacillus. 

The examination of the stools for tubercle bacilli is not always satis- 
factory. The enormous number of bacteria of the feces may prevent a recog- 
nition of the tubercle bacillus even though it is present. In the feces we find 
certain organisms which are acid-fast, such as the timothy bacillus, which is 

1 This sugar-free broth is specially prepared to remove the muscle-sugar. Meat extract 
is inoculated with colon bacilli and incubated for 16 hours. Boil and strain through cloth. 
The nitrate is then made up as ordinary bouillon. 

2 Or the colonies may be transferred to Russell's medium (litmus-lactose-glucose agar) as 
recommended by Lumsden (Pub. Health Reports, 1912, XXVII, 7S9). 



THE FECES. I33 

very closely related in morphology and staining characteristics to the tubercle 
bacillus. We should be on our guard, therefore, lest we make a wrong diagnosis. 

If these organisms are present on repeated examination and there are clinical 
symptoms pointing to such a trouble of the bowel, one may give a presumptive 
diagnosis of tuberculosis, remembering that the tubercle bacilli may have 
come from swallowed tubercular sputum. 1 

In selecting the material for examination, pick out the particles of mucus, 
especially those which are blood-stained or purulent. One may be much 
more certain of definite results if the antiformin or Petroff method be used upon 
the feces, which has been rubbed up with distilled water (see p. 18). The 
staining methods are as usual. 

The Bacillus aerogenes capsulatus (Bacterium Welchii; gas bacillus) is fast 
coming into prominence as an etiologic factor in many cases of intestinal dis- 
turbance associated with diarrhea, although it is possibly a normal habitant of 
the intestinal tract of children. 2 

VI. Parasitology of the Feces. 

In the examination of the feces for parasites one should obtain the feces 
as fresh as possible. This is especially the case where the examination for 
protozoa such as the ameba is to be made. The feces should be kept in a 
warm vessel prior to the examination, as the motility of these unicellular organisms 
is shown only with great difficulty after they have become cold. . The other 
types of protozoa are less sensitive to changes in temperature and show their 
active motility, providing the feces be examined soon after voiding. It seems to 
be generally accepted that these organisms are the more easily found the more 
fluid, more mucoid, and more alkaline the feces. The particles for examination 
should be preferably the masses of mucus which can be found by careful search 
in the liquid stool. The organisms are best examined in their fresh condition, 
as the staining agents usually require fixation of the specimen and consequent 
death of the parasite. 3 

In the examination of the feces for parasitic ova the following method of 
Yaoita (Deutsch. med. Wchnschr., 1912, XXXVIII, 1540) is, in my opinion, 
the best. From several different parts of the fecal mass take portions the size 
of a pea. Place in a test-tube and add 5 c.c. of 25 per cent, antiformin solution. 
Mix thoroughly and warm but do not boil. Allow to cool, add 5 c.c. of ether 
and shake well. Filter the mixture through one layer of gauze and centrifuge 
the filtrate for one minute. Four layers are formed, in the lower of which are 
the ova along with the resistant cellulose, connective tissue and muscle fibers 
and salts. Pour off the supernatant fluid and treat the sediment with dilute 

1 See Laird, Kite and Stewart, Jour. Med. Research, 1913, XXLX, 31; Bergstrand, Hygiea, 
1915, LXXVII, 97; Petroff, Jour. Exper. Med., 1915, XXI, 38. 

2 See Kendall and Smith, Boston Med. and Surg. Jour., 1911, CLXIV, 306; Kendall, 
Ibid., 1912, CLXV, 75; Orton, Jour. Med. Research, 1913, XXIX, 287; Knox and Ford, Bull. 
Johns Hopkins Hosp., 1915, XXVI, 27; Morse, Am. Jour. Med. Sc, 1915, CXLIX, 17. 

3 Ross (Further Researches into Induced Cell-reproduction and Cancer, Philadelphia, 19 n 
and 19 1 2) has advocated the method of embedding the suspected feces in agar-jelly which may 
be impregnated with the stain. See, also, Smithies, Arch. Int. Med., 1912, IX, 736. 



134 DIAGNOSTIC METHODS. 

HC1 and a little ether. Shake well and centrifuge again. This latter process 
is repeated two or three times. The ova are thus freed from most of the other 
fecal material and may be detected readily by microscopic examination of the 
sediment. 1 
(i). Protozoa. 

The protozoa are unicellular animal organisms. These, although living 
occasionally symbiotically, are more usually found as isolated single organisms. 
A few of these organisms are sufficiently large to be detected by the naked 
eye, but the majority are minute and require the finer microscopic detection. 
They consist essentially of a mass of protoplasm (cytoplasm or sarcode), 
with differentiation for functional purposes (organelles) of a variable character, 
constancy, and prominence. In the ameba, for instance, the sarcode may be 
separable into an internal distinctly granular portion known as the endosarc, 
and a peripheral clearer portion known as the ectosarc; a cell-membrane in 
some instances is a well-marked feature, while in others it is absent; and 
in some of the free-living protozoa special external coverings of chitinous, 
siliceous, or chalky composition enclose the protozoon. Of the various parts 
the nucleus is, after the cytoplasm, the most constant, varying much in appear- 
ance, shape, size and number in the individual form (single nucleus of variable 
size and shape; double or dimorphic nucleus, a macronucleus of vegetal 
character, a micronucleus with creative function; polymorphous nucleus, 
multiple nuclear granules more or less widely distributed in the cytoplasm). 

Not uncommon examples of specialization are met in the contractile vacu- 
oles, in pigment spots, in mouth-like ingestion foci and their pits on the surface 
of many forms with relatively firm cell membrane, in the anus-like excretory 
points of the same form, or the peripheral motor organelles, in the sucking 
tubes of the suctoria, and in the hook-like fixation apparatus of the gregarines. 

Motile protozoa move in a variety of ways. The naked rhizopods move 
by a peculiar rolling due to currents in the internal substance of the cell or by 
the protrusion of the cell-substance as extensions or pseudopoda, these move- 
ments being always accompanied by change in the cellular shape of the animal. 
Ciliates and flagellates move through the activity of the special cuticular 
appendages known as ciliae and flagella. 
(a). Rhizopoda (Sarcodina). 

Amoebina. 

(a). Amoeba coli (endamceba histolytica). 

The ameba was first discovered in the large intestine by Lambl, although 
we are indebted to Losch for the first accurate description of this organism. 
He did not believe it to be the cause of dysentery, but regarded it as a secondary 
invader. Since that time much work has been done, a controversy having 
arisen regarding its specificity. In a great many cases of dysentery this organism 
is not found, but in its stead bacteria, especially the Shiga bacillus, are present in 
large numbers. This bacillary dysentery is something entirely different from 
the amebic type of this disease. It is true that in certain cases of dysentery as 
well as in normal individuals, amebae are found which are differentiated with 
1 See Faun tier oy and Hay den, U. S. Naval Med. Bull., 19 15, IX, Si. 



THE FECES. 135 

great difficulty from the true amceba coli. There seem to be two distinct types: 
one pathogenic, to which Councilman and Lafleur give the name amceba dysen- 
teric, which Losch styles amceba coli, and which Schaudinn designates end- 
amoeba histolytica ; a second type, which is non-pathogenic, has been styled by the 
first writers amceba coli Losch and by Schaudinn endamceba coli. Viereck 1 has 
described a third form, which is pathogenic and is known as the endamceba 
tetragena. This form is now believed to be identical with the endamceba histo- 
lytica. These forms have not been cultivated. The cultivated forms are non- 
pathogenic to man. 2 



G 







Fig. 24. — Amoeba coli. (Hemmeter.) 

One may sum up the points relative to the pathogenicity of the endamceba 
histolytica as follows : (1) It appears in a form of dysentery which is anatom- 
ically characterized by peculiar ulcerations which are markedly different from 
the diphtheritic inflammatory processes of the bacillary dysentery. (2) The 
more recent the case the more numerous are these parasites; (3) they are depos- 
ited in the dysenteric ulcers and tend to pass into the deeper tissue appearing 
as true tissue parasites; (4) they frequently deposit themselves in the liver caus- 
ing abscesses which contain these organisms in large numbers and practically no 
other infectious material; (5) by injection of amebae-containing feces into the 
large intestine of animals typical amebic dysentery may be caused. It is to be 

1 Beiheft z. Arch. f. Schiffs. -u. Tropenhyg., 1907, I, 1. 

2 See Craig, Arch. Int. Med., 1911,-VFI, 362; Am. Jour. Med. Sc, 1912, CXLV, 83; Jour. 
Med. Research, 1912, XXVI, 1; War Dept., Office of Surgeon General, Bull. 2, 1913, 95; Jour. 
Am. Med. Assn., 1913, LX, 1353; South. Med. Jour., 1913, VI, 370; Jour. Infect. Dis., 1913, 
XIII, 30; Whitmore, Arch. Int. Med., 1912, FX, 515; Darling, Arch. Int. Med., 1913, XI, 1 
and 495; Jour. Am. Med. Assn., 1913, LX, 1220; James, New York Med. Jour., 1913, XCVHI, 
702; Couret and Walker, Jour. Exper. Med., 1913, XVIII, 252; Williams and Calkins, Jour. 
Med. Research, 1913, XXIX, 43; Giffin, Jour. Am. Med. Assn., 1913, LXI, 675; Walker 
and Sellards, Editorial, Jour. Am. Med. Assn., 1914, LXII, 300; James, Ann. Trop. Med. and 
Parasitol., 1914, VIII, 133; Craig, Arch. Int. Med., 1914, XIII, 737 and 917; Sellards and 
Baetjer, Am. Jour. Trop. Dis. and Prev. Med., 1914, II, 231; Bull. Johns Hopkins Hosp.. 
1915, XXVI, 45; Couret, Am. Jour. Trop. Dis. and Prev. Med., 1915, II, 450- 



i 3 6 



DIAGNOSTIC METHODS. 



borne in mind that symbiosis with bacteria is apparently necessary for the 
development of amoebae. 

The endamceba histolytica is an actively motile, roundish, pear-shaped, 
oval, or irregular unicellular organism having an endosarc which is typically 
granular and may contain leucocytes, red blood- cells, bacteria, particles of 
food, or pigments which the parasite has ingested, and shows the clear hyalin 
ectosarc, which is, perhaps, best seen in the pseudopoda. The pseudo- 
poda are the typical motile portions of the parasites, these projections being 
thrown out from any point of the periphery, the protoplasm seeming to flow 
into them and drawing the animal after it. The parasite may not move, but 
will change its external appearance by throwing out these pseudopoda in various 
directions. The nucleus is a homogeneous, little refractile, chromatin-poor, 








Fig. 25. — Coccidium hominis, from intestine of rabbit: 1, A degenerate epithelial cell 
containing two coccidia; 2, free coccidium from intestinal contents; 3, coccidium with four 
spores and residual substance; 4, an isolated spore; 5, spore snowing the two falciform 
bodies — X n 40. {Tyson after Railliet.) 

spherical mass, about 6 microns in diameter (the diameter of the organism itself 
ranging between 10 and 50 microns, the average being 35). This nucleus is 
not always clearly visible, appearing much more frequently in the animal killed 
by corrosive sublimate. In the granular endosarc one frequently sees several 
vacuoles which may or may not pulsate, the general opinion being that 
pulsation is absent, although change in shape is frequent. 1 

The stools of this amebic dysentery are thin and watery, show an alkaline 
reaction, and have a peculiar lime-like odor. Much mucus, blood, and occa- 

1 If one wishes to stain such preparations, the method of Darling (Jour. Am. Med. Assn., 
1912, LIX, 292, and Science, 19 13, XXXVII, 58) is reliable. Stain with Wright's stain in 
the usual way and follow this with Giemsa's stain until the film has a purple cast. Then plunge 
this into 60 per cent, alcohol to which 10 to 20 drops of aqua ammonias have been added. Dif- 
ferentiate in this way until the film has a violet color. Dry and examine. See, also, Craig, 
War Dept., Office of Surgeon General, Bull. 1, 1913, ri. 



THE FECES. I37 

sionally many pus-cells are found for which reason the mucus should be 
selected for examination in case these organisms are suspected. 1 Frequently 
one may obtain better specimens for examination by examining mucus which 
is obtained with a rectal tube. 

(/?). Endamoeba coli (amoeba coli Losch). 

This parasite varies in size between 10 and 15 microns. The hyaline 
protoplasm of the pseudopoda is not distinctly differentiated from the ecto- 
plasm. It is opaque, gray in color, and its nucleus is sharply defined, being 
characterized by its richness in chromatin. The movements of this organism 
are not as rapid as those of the endamoeba histolytica, which does not show as 
active phagocytic power. According to Craig, about 65 per cent, of normal 
persons show these non-pathogenic endamoeba coli in the feces. 
(b). Sporozoa 

Coccidium hominis (coccidium perforans; cystospermium hominis). 

This organism appears in the feces as an oval or spherical parasite about 
22 microns long and showing a thin periphery. A large number of nuclei are 
usually observed. 

The infection with these organisms seems to arise from rabbits in whose 
intestines these parasites develop in large numbers. 
(c). Flagellates. 

(a). Trichomonas intestinalis. 

This organism was first studied by Marchand and Zunker and later 
elaborated by Grassi, Roos, and Janowski. It is probably identical with the 
one known as trichomonas vaginalis which may live in the 
vagina, the urethra, large and small intestine, the stomach, 
and may be found in the sputum. Various forms have been 
described as being found in the intestine, but they are in all 
probability the same organism; among these we find protoryxo- 
myces coprinarius, monocercomonas hominis, cimcenomonas homi- 
nis, trichomonas hominis, cercomonas coli hominis, and cerco- 
monas seu Bodo urinarius. 

This is a colorless protozoon of a pyriform or spindle 
shape, rounded in front and bearing three flagella which are 
apt to be merged at the base and easily lost, the posterior end 
pointed but not bearing a flagellum. It is from 20 to 25 
microns in length and 8 to 12 broad. Along the body, starting 
from the base of the flagella, runs an undulating membrane in Trichomonas 
a somewhat spiral manner to the posterior end. It has a finely intestinalis. 
granular cytoplasm and at its anterior end a vesicular nucleus, ^ v son -) 
behind which one or more non-pulsating vacuoles may be seen. At times 
this organism may be observed to assume an ameboid form, the movements 
of the flagella having then ceased and projections resembling pseudopoda 
being observed. 2 

1 Musgrave and Clegg advise the administration of a saline cathartic and the later ex- 
amination of the fluid portion of the stool. 

2 See Castellani, Jour. Trop. Med. and Hyg., 1914, XVII, 65; Lynch, New York Med. Jour., 
1915, CI, 886. 




138 



DIAGNOSTIC METHODS. 



(0). Cercomonas hominis. 

This organism was first studied by Davine and has been found by many 
other workers. It is known under the names of cercomonas intestinalis, mono- 
cercomonas hominis, cimccnomonas hominis. The adult organism is a small, 
colorless, pyriform parasite, with round anterior end provided with one long 
flagellum and a pointed posterior end. It is 8 to 10 microns long and has no 
undulatory membrane, as has the trichomonas intestinalis. 




"^^V 



Fig. 27. — Cercomonas hominis 
A, larger and B, smaller varieties 
(Tyson.) 



(7). Megastoma entericum. 
This organism was first found by Lambl 
in the feces of children. It is known under 
several names among which are Lamblia in- 
testinalis, hexamitus duodenalis, dimorphous 
muris, and megastoma intestinale. This parasite 
is a colorless pear-shaped protozoon with a rounded anterior end and a pointed 
posterior end bearing a pair of flagella. The anterior end has one side concave 
with a raised border or lip, one pair of flagella, arising at the anterior border 
of this disk-like concavity, and two pairs together from its posterior margin. 
The cytoplasm is finely granular and the dumb-bell shaped nucleus is 
anteriorly about the middle level of the concavity. Vacuoles are absent and 
solid inclusions are never observed. The length of these organisms is from 
15 to 16.5 microns, while the width is from 10 to 12.5 microns. The number 
of these organisms found in the feces may be very large. The surest points 
for their diagnosis seem to be the concavity and the dumb-bell shaped nucleus. 
The source of infection for man is the drinking of infected water. 




(d). Infusoria. 

Balantidium coli (paramoecium 
coli). 

This parasite is colorless, ovoid 
in shape, 70 to 100 microns long and 
50 to 70 microns broad, having a finely 
granular cytoplasm containing frag- 
ments taken from the intestinal ma- 
terial, and a clear ectoplasm showing 
numerous longitudinal striations. It 
is covered completely with actively 
motile cilia which are more dense 
about the funnel-shaped mouth which 
extends about one-fourth the length of the body. The nucleus is kidney 
shaped and is usually accompanied by one or more accessory nuclei, while two 
or more contractile vacuoles are seen which pulsate to a slight extent. 

This parasite has been found in connection with various types of diarrheal 
affection and also in persons entirely free from intestinal symptoms. It may 
be present in the stools in very large numbers, being found especially in the 



Fig. 28. — Megastoma entericum, showing 
disk-surface and lateral views in larger 
figures, and three epithelial cells with 
attached examples to the right. (Tyson.) 



THE FECES. 



139 



colon, but in severe cases also in the jejunum. The hog is the most common 
host of this parasite, which at times shows marked pathogenicity (see Bowman 1 ). 

(2). Entozoa (Enthelmintha). 
(4). Platodes (Flat Worms), 
(a). Cestodes (Tape -worms). 

The cestodes are naked, flat worms of elongated ribbon shape, endopara- 
sitic, at least in their adult stage, and in many instances in all stages, without a 
digestive canal, and always more or less distinctly divided into segments. The 
entire parasite, or strobile, may be looked upon as a colony of individuals 
united in ribbon fashion from their mode of origin, for convenience in their 
development and functional performance; the various segments being derived 
by a process of constriction from the originally acquired parasite, which is spoken 
of as the head, nurse, or scolex of the strobile. A characteristic of the cestodes 
is the differentiation of two developmental stages: the first, cysticercus stage, 
in which the connective tissue or parenchymatous organs are invaded, and, 
secondly, the development of the sexually mature 
animal in the intestine. The scolex obtains en- 
trance as a larva into the intestinal tract of the host, 
becomes attached by a special fixation apparatus to 
the mucous membrane, and there develops into the 
adult parasite, forming the anterior extremity of the 
strobile in the developed worm. The head is usu- 
ally a very small and inconspicuous object of globular, 
pyriform or club shape with a short posterior exten- 
sion, spoken of as the neck. In the middle of the 
frontal face of the head there is often a small promi- 
nence, known as the rostellum, about which may be 
arranged in one or more rows, as one of the means 
of fixation of the parasite, small hooklets as a crown. 
As more constant means of fixation the head is 
provided with two or four suckers, rounded or linear depressions with more 
or less definite lips. Back of the head by a process of constriction from the 
neck, the segments, also known as links or proglottids, arise, the newest form 
always being placed between the neck and the next older link. Thus the older 
segments are always separated more and more from the head by each newly 
formed proglottid, each as it grows older and recedes further from the neck 
developing in size; the length of the strobile being thus dependent upon the two 
factors, growth of the individual length and the new formation of segments. 
These new segments as they are first formed are usually very short and pro- 
portionately broader, but as they increase in size with age they generally enlarge 
especially in their long diameter, and come to be more or less square. The 
number of these links may vary from three or four to several thousands, the 
length of some worms being 100 or more feet. The structure of each link and 
hence of the whole strobile includes an interior or matrix of an indeterminate 

1 Jour. Am. Med. Assn., 1911, LVII, 1814. See, also, Hinkelmann (New York Med. Jour., 
1 91 5, CI, 200), who finds these parasites present in water supplies. 




Fig. 29. — Balantidium coli: 
a, Nucleus; b, vacuoles; 
c, cytostome, with pit and 
peristome; d, ingested 
material. (Tyson after 
Leuckart.) 



140 DIAGNOSTIC METHODS. 

connective reticular material, from which the various organs appear to develop 
and in which they are imbedded; over which are to be recognized exteriorly a 
delicate cuticle and beneath the latter, two layers of so-called muscle, the outer 
layer longitudinal and the inner transverse and circular. 1 

Aside from the common parts the various links may be looked upon as 
individuals. There is no digestive canal, all nutrition being obtained by the 
parasite from absorption of dissolved material from the fluids in the infested 
intestine. The only really highly organized parts are the generative organs, 
each link containing both male and female organs. The terminal links are 
the ones containing the most ova, while the links nearest the head are usually 
but partially developed. In their development the ova fill the canal of the 
oviduct more and more often causing the appearance of side pockets of more 
or less branching character. The terminal ripe links either actually containing 
the ova or, after discharge of more or less of the original, number, are apt to 
become separated from the strobile and be carried with the fecal matter from 
the intestine. Either with or without intermediate development the embryo 
is in some way, by water or in solids, carried into the alimentary canal of a 

second host. Arrived in this situation, both by its 
own activity and by passive convection by blood 
and lymph streams, the embryo penetrates the 
intestinal wall and becomes deposited in one or 
other situation as a larva, bladder- worm, or cysti- 
cercus. This larval or cysticercus form is sur- 
rounded by a delicate connective tissue outer wall, 
derived from the host by a process of reactive in- 
flammation, within which lies the true bladder- 
worm. This is essentially the head of the future 
parasite. This cysticercus, after remaining a vari- 

„* , , , , able period in the tissues, is devoured with the 
Fig. 30. — Head and neck, and r ... 

ovum x 300, of taenia solium, flesh of its host by a third (definitive) host. 
Embryophore surrounded by / \ t "a 

vitellus. {Tyson after Gould.) ( a )- l«nil<iae. 

(1). Taenia solium. 
This parasite, which is derived from infection through the cysticerc 
cellulosae of pork, has been also named tcenia cucurbitina, tccnia dentata, 
cystotcenia solium, and pork tape-worm. The average length of the strobile 
is 2 to 3 meters, occasionally reaching twice this measurement. The head 
is somewhat spherical or slightly tetragonal from the four rather prominent 
cup-like suckers with thick lips. The head varies from 1/2 to 1 mm. in di- 
ameter, while the suckers range from 1/4 to 1/2 mm. in diameter. It is pro- 
vided with a short, thick, rostellum bearing a double crown of hooklets, usu- 
ally 28 in number. The neck is thin, about 3 cm. in length and is unsegmented. 
The proglottids number between 800 and 900, the fully grown and ripe seg- 
ments measuring from 9 to 12 mm. long and 5 to 6 mm. broad. The uterus 
consists of a large, median, longitudinal trunk with from 7 to 10 coarsely den- 

1 See Douthitt, 111. Biological Monographs, 191 5. I, No. 3. 





he 

. 

to. 



THE FECES. 



141 



dritic branches on each side. The ova are round or oval, the shell very thin 
but surrounded by an embryonic layer which is thick and shows distinct radiat- 
ing lines. These eggs are usually of a brownish color and may show on their 
interior the hooklets of the embryo. 

This parasite in its adult stage is practically limited to the small intestine 
of man, while the larval form has been found in swine, monkeys, dogs, etc. 
It has been shown that careful cooking and prolonged and thorough salting and 
drying of the meat will destroy the vitality of the cysticercus cellulosae. It 
is, therefore, plain that any meat should be more or less thoroughly cooked 
before being eaten. 

(2). Taenia saginata. 
This parasite is the beef tape-worm and is also known as the tamia medio- 
canellata, tcenia inermis, and tcenia dentata. The adult worm varies from 3 
to 8 meters in length, has a head from 1 to 2 mm. in diameter, tetragonal in 
shape without hooklets or rostellum, with four cup-shaped suckers each 0.8 
mm. in diameter and placed at the corner of the frontal face. The ripe seg- 
ments are from 18 to 20 mm. long and 5 to 7 mm. broad. The uterus shows 
a distinct median longitudinal trunk with 20 to 35 lateral single or dichotomously 
branching and slender diverticula. The eggs are spherical with a thin shell 
surrounded by a thick radially striated embryonic shell. These eggs are from 
30 to 40 microns long by 20 to 30 microns wide. 
(3). Taenia cucumerina. 
Infection with this parasite is relatively rare in the United States. It is 
found almost exclusively in children, the infection occurring through dogs and 
cats, the larval form of the parasites being found in 
the body lice and fleas. 1 This organism has other 
synonyms, among them being tcenia canina, tcenia 
moniliformis, tcenia elliptica, dipylidium 
caninum, and dipylidium cucumerinum. 
The parasite is from 15 to 35 cm. in 
length. The head is small, rhomboidal, 
with a clavate rostellum surrounded by 
three or four crowns of hooklets (48 to 60 
in number), suckers rather large with 
radially marked borders, and neck very 
short. The segments are from 80 to 150 
in number, the older ones being 8 to 11 
mm. in length and 1 to 3 mm. in breadth 
Fig. 31.— Head and and often showing a reddish-brown color. 
The links frequently swell out in the 
middle so that the parasite has an ap- 
pearance not unlike a chain of beads. 
A single uterus is common to the two oviducts, consisting of a network of tubes 
in which the ova lie in groups, filling small saccules, each containing 10 or 15 
ova and surrounded by a reddish material which gives the color to the worm. 

1 See Lins, Wien. klin. Wchnschr., 1911, XXTV, 1595. 





neck of taenia saginata : A, 
retracted; B, extended. 
[Tyson after Gould.) 



Fig. 32. 
Taenia cu- 
cumerina. 
{Tyson after 
Leuckart.) 



142 



DIAGNOSTIC METHODS. 



The ova are spherical, 43 to 50 microns in diameter, and have a double wall. 
Within the wall one observes an embryo armed with hooklets. 

(4). Taenia nana. 

This worm is known as the " dwarf tape-worm" and is perhaps best known 
in Italy and Southern Europe, although it has been found in many cases in the 




Fig. t>?> • — Taenia nana: X 10. {Tyson after Gould.) 

eastern and southern portions of the United States. It has been called the 
tenia (Egyptica, hymenolepis nana, hymenolepis murina, and diplacanthus nana. 
The infection with the ova is probably through the use of unrlltered water tainted 
with human or murine dejecta. It is most frequently seen in children, and 
inhabits the ileum, usually from the middle toward the ileocecal valve. 

The parasite is from 10 to 15 mm. in length and 
from 0.5 to 0.7 mm. broad, is provided with a subglobu- 
lar head measuring 0.2 to 0.3 mm. in transverse di- 
ameter, shows four large rounded suckers and a large 
rostellum retractile into an infundibulum. The rostellum 
is surrounded by a single row of characteristic hooklets, 
24 to 30 in number and 14 to 18 microns in length. The 
neck is rather long and slender, being followed by about 
150 small proglottids, which are broader than long (0.4 to 
0.9 mm. broad by 0.14 to 0.3 long). 

The ova are characteristic. They 
are round or oval in shape, 32 to 36 
by 42 to 56 microns in size, and have 
two distinct membranes. At each 
pole of the inner membrane is seen a 
small protuberance from which springs 
a number of clear refractile threads, 
which are distributed in a waving 
fashion through the substance interme- 
diate to the outer and inner walls. 




Fig. 34.— Head 
and neck of taenia 
diminuta. {Tyson 
after Braun.) 




Fig. 35. — Ovum 
of taenia diminuta. 
{Tyson after Braun.) 



(5). Taenia diminuta. 

Synonyms. — Hymenolepis diminuta; hymenolepis flavopunctata; tenia 
leptocephala; tenia flavopunctata; tenia minima; and tenia varerina. 

The parasite is 10 to 60 mm. long, head small, globular, with four globose 
suckers situated close to apex, rostellum small, pyriform, and devoid of 
hooklets. The segments number 800 to 1300 and are broader than long. 
Gravid uterus nearly fills the segments, showing as transverse line when not 
ripe. 



THE FECES. 



143 



The ova are round or slightly oval, yellowish in color, double-walled, inner 
wall showing slight protuberances at poles, a layer of albuminous material 
being seen between the walls. 

(6). Taenia echinococcus. 

This parasite in its adult stage is met with in the upper part of the small 
intestine of dogs, wolves, and jackals. The larval form, known as the hydatid 
cyst, is found in man, although more frequently in the ox, hog, horse, dog, 
cat, rabbit, etc. This disease is rare in America, and is acquired through 
association with the dog more frequently than in other ways. The most 
common seat of hydatid disease is in the liver. 1 




Fig. 36. — Taenia echinococcus: 
a, Adult; &, head from echinococcus 
cyst. On left a detached hooklet, 
as seen in fluid from cyst. (Tyson 
after Coplin and Bevan.) 



Fig. 37. — Hydatid cyst, showing daughter cysts. In 
the lower part of field is a whitish mass containing 
parts of the walls of ruptured daughter cysts. The 
thick wall of the mother cyst is well shown. From 
liver of man, X f . (Coplin.) 



The parasite is 2 to 5 mm. long, has a small subglobular head measuring 
0.3 mm. in transverse diameter and bearing a rostellum with a double row.of 
very characteristic hooklets (28 to 50 in number) and four prominent cup- 
shaped suckers. The neck is short and rather thick; proglottids three or four 
in number, the last of which is usually longer than the rest of the worm put 
together. Uterus consists of a thick longitudinal median trunk with a few 
short lateral branches. 

Ova spheroidal with thin radially striated shells and containing a granular 
hexacanthus embryo. Length of ova 30 to 36 microns, transverse diameter 
25 to 30 microns. 

1 See Phillips (Jour. Am. Med. Assn., 1913, LXI, 1981), who reports such a cyst in the 
pancreas. 



144 



DIAGNOSTIC METHODS. 



dibothrium latum; 



(b). Bothriocephaloidea. 
(i). Bothriocephalus latus. 

Synonyms. — Dibothriocephalus latus; taenia lata; 
bothriocephalus latissimus; fish tape-worm. 

This parasite is most commonly met in the human intestine, but may be 
found in dogs and cats. It is most common in central Europe and in the 
maritime countries of Europe, British Islands, and Japan. The examples 
found in America occur in foreigners, as a rule. The ova, which are usually 
in large numbers in the feces, re- 
quire for their further development 
immersion in water. The liberated 
embryo is then taken up by fresh- 
water fish and conveyed to man. 

Strobile 2 to 10 meters (20 in a 
few cases) in length, marked in ripe 
segments by brownish central rosette 
(uterus with ova). Head elongated, 
almond shaped, 2 to 5 mm. long 
and 0.7 mm. transversely, with two 
lateral grooves or bothridia as suck- 
ers. Neck variable according to de- 
gree of contraction. The segments 
number 3,000 to 4,000 and begin 
about 50 cm. from the head. The 
anterior links are poorly defined, in 
their growth increasing slowly in 
length but markedly in breadth. 
The ripe links measure 2 to 4 mm. 
in length and 10 to 12 in width, 
with opaque brownish rosettes in 
the middle line. Uterus formed of 
a number of plicated tubes in the 
form of a rosette. 

The ova are brownish in color, ellipsoidal in shape, 68 to 71 microns in 
length and 44 to 45 in transverse diameter, have a thin shell, and a lid 
which may be opened or closed. The contents of the ova are coarsely granular 
or mulberry-like. 

Infection with this worm is not always single, as high as 100 worms having 
been reported in the same individual. Many cases of infection with this para- 
site are associated with a high-grade anemia, which is distinguishable from 
pernicious anemia only by the effects of removal of the parasite. 

(2). Dibothriocephalus cordatus. 
This is a tape-worm of the same genus as the above and is parasitic in 
seals, being transmitted from them to man. It varies in length from 80 to 
115 cm. Proglottids about 600, 7 to 8 mm. broad. The head is heart- 




Fig. 38. — Bothrio- 
cephalus latus. (Tyson 
after Leuckart.) 



Fig. 39.— Diboth- 
riocephalus cor- 
datus: adult. (Tyson 
after Leuckart) 



THE FECES. 



!45 



shaped, 2 mm. long and broad. The ova are 
similar to those of the latus, but are a little 
larger in size. 

(3). Bothriocephalus sp. Ijima et 
Kurimoto. 

Synonyms. — Diplogonoporus grandis; Krab- 
bea grandis. 

Strobile measures up to 10 meters in length. 
Proglottids short and broad, head, neck, and num- 
ber of segments unknown. Uterus rosette-shaped 
with several loops on each side. Ova brownish, 
operculated, oval, 63 microns in length and 48 to 
50 in width. Intermediate host unknown, prob- 
ably fish. 

(0). Trematodes (Fluke-worms). 

The various forms of distoma, which belong 
to this class, are more properly hepatic parasites, 
although they and their ova may at times appear 
in the intestines and feces. A detailed discussion 
of these parasites will be taken up in the chapter 
on Parasites. 

(B) . Nematodes (Round Worms) . 
The nematode worms are unsegmented, 
elongate, circular or nearly so in their transverse 
section, cylindrical or more or less delicately 
fusiform and tapering toward each end. They 
are with but few exceptions parasitic and include 
many important examples, which are parasitic in 
man. Intermediate hosts, so essential for the 
intermediate development of the flukes and tape- 
worms, are practically absent in the nematodes. 



a* 



- 



(a). Ascaridae. 
(1). Ascaris lumbricoides. 

This is the common round worm or maw- 
worm seen so frequently in children. The number 
in a single host is usually small, but may be 
very large. Its habitat is the small intestine, 
but the eggs may occur in the vomitus as well 
as in the feces. 

The male worm is whitish to reddish-yellow 
in color; 15 to 17 cm. long, 3 to 3.5 mm. thick; elongate, fusiform; cuticle 
finely ringed; oral orifice terminal with three lips (one dorsal and the other 
two meeting in the median ventral line), each with fine denticulations on 



Fig. 40. — Ascaris lumbri- 
coides: to left, male in lateral 
aspect; to right, female, ventral 
aspect, natural size. {Tyson 
after Railliet.) 



146 



DIAGNOSTIC METHODS. 



margins; at base of superior lip two papillae, one only at base of other two 
lips; posterior end terminating conically, curved ventrally, with two slightly 
curved, short, equal spicules projecting from the subventral cloaca; 70 to 
75 papillae on the ventral face of posterior end, of which seven pairs are 
postanal. 

The female parasite is 20 to 25 cm. long, 5 to 5.5 mm. thick; anterior end 
and general appearance as in male; posterior end tapering, ending in a conical, 
pointed, straight tail, vulva at level of first third of body length (in a slightly 
depressed annular band) ; anus subterminal. 

The ova are elliptical in shape, 50 to 75 microns long and 40 to 58 
microns broad; shell thick and transparent; stained yellowish with fecal 
material when in feces, but colorless in uterus; protoplasm unsegmented 
and coarsely granular; covered with a mammilated albuminous envelope 
which may be lost. x 




Fig. 41. — Ascaris mystax. 
A, Male; B, female; C, anterior extremity, enlarged and shown from dorsum to exhibit 
the lateral wing-like cuticular expansions; D, same showing in profile. {Tyson after 
Railliet.) 



(2). Ascaris mystax. 

Synonyms.— Ascaris canis; ascaris lumbricus canis; ascaris teres; ascaris 
caniculce; ascaris cati; ascaris tricuspidata; ascaris felis; ascaris werneri; ascaris 
marginata; ascaris alata; suidfusaria mystax. 

The male parasite is whitish or slightly brownish; 40 to 60 mm. long, 1 
mm. thick; anterior end usually curved, with lateral cuticular expansions 
making the end look somewhat arrow-like; mouth terminal with three nearly 
equal lips with denticulate margins; at base of superior lip two papillae, on 
inferior lip one ordinary and two minute papillae; posterior end curled and with 
lateral cuticular alar expansions, and on each side of cloacal aperture 26 
papillae, of which five are postanal. 

1 See Foster, Jour. Parasitol., 1914, I, 31. 



THE EECES. 



147 



The female worm is 120 to 180 mm. long; anterior end and general 
appearance as in male; posterior end straight, terminating obtusely; vulva 
at anterior fourth of body length; anus subterminal. 

The ova are almost spherical; 68 to 72 microns O. 

in diameter; shell thin with thin albuminous envel- 
ope showing an alveolated surface. 1 

(3). Oxyuris vermicularis. 

Synonyms. — Ascaris vermicularis; fusaria ver- 
micularis; ascaris grcecorum; pin-worm; thread-worm; 
seat-worm. 

The male is whitish in color; 3 to 5 mm. long, 
0.3 to 0.4 mm. thick; cuticle transversely striated 
and at head end showing a vesicular swelling along 
the dorsal and ventral median lines; lateral lines 
distinct; mouth terminal, with three retractile lips; 
esophagus with distinct bulb; posterior end conical, 
curved ventrally, with six pairs of papillae and slight 
cuticular expansion on each side; one spicule hooked 
at free end. 

The female is 10 mm. long, 0.6 mm. thick; 
anterior end and general appearance as in male; 
posterior end straight, extended to a long mucronate 
tail; vulva at anterior third of body length. 

The ova are oval, flattened on one side with a 
characteristic asymmetry, 50 microns long and 16 to 
20 broad; shell thin; colorless. 

This parasite inhabits the rectum and colon, 
but it may travel even into the stomach. The ova 
are rarely found in the feces, except in the mucus or 
about the anus. 2 




Fig. 42. — Oxyuris vermi- 
cularis: to left, female; to 
right, male (considerably 
enlarged); A, anus; O, 
mouth; v, vulva. (Tyson 
after Braun.) 



(b). Angiostomidae. 
Strongyloides intestinalis. 

Synonyms. — Anguillula intestinalis et stercoralis; 
leptodera intestinalis et stercoralis; pseudorhabditis 
stercoralis ; rhabdonema strongyloides; rhabdonema 
intestinalis. 

This organism is found in two different forms: the first dioic and free, 
the second parasitic, as parthogenetic females. The parasitic form lives in 
the upper intestinal tract of man; is 2.5 mm. long, cylindrical, with pointed 
tail end, cuticle smooth; mouth simple with four lips; long, slender, cylin- 
drical esophagus reaching one-fourth of the length of the worm; anus close 

1 See Beisele, Munch, med. Wchnschr., 1911, LVIII, 2391; Flury, Arch. f. exper. Path. u. 
PharmakoL, 1913, LXVII, 294. 

2 See Crowell and Hammack, Philippine Jour. Sc, 1913, VIII (B), 157; Willets, Ibid., 
1914, IX, 233; Fracker, Jour. Parasitol., 1914, 1, 22; Aschoff, Berl. klin. Wchnschr., 1914, LI, 
1504. 



148 



DIAGNOSTIC METHODS. 



to tail; vulva at posterior third, containing yellowish-green oval ova, 50 to 58 
microns long and 30 to 34 microns broad. The larvae develop in the intestine 
and are passed in the fecal material. These larvae are at first from 200 to 
240 microns in length, but increase to two or three times this length. The 
larvae differ essentially from the parent in having a rhabditiform esophagus. 
In the discharged feces at about 30 C. these develop with one moulting of 
the cuticle to a free-living generation with separate sexes. 




Fig. 43. — Strongyloides intestinalis; on the left, a gravid female from human intestine 
(natural size 2.5 mm.). In the middle, a rhabditiform. larva from fresh fecal matter, X 120; 
to the right, a filariform larva from culture, X 120. {Tyson after Braun.) 



In this free sexual generation the worms are smooth, cylindrical, and 
tapering, with pointed tail-ends; the mouth is the same as in the parasitic 
form; esophagus rhabditiform with its anterior portion long and with the pos- 
terior pyriform and containing a Y-shaped chitinous armature; anus at base 
of tail; male with tail curved and two spicules, body length 0.7 mm.; female 
1 mm. long, with straight pointed tail, vulva a little back of the middle; ova 
few, yellowish, ellipsoid, thin-shelled, 70 by 45 microns in size, sometimes 
hatching in the uterus. The larvae of this generation look much as their 



THE FECES. 1 49 

free parents, are at first 0.22 mm. in length, but grow to 0.55 mm., then moult 
and assume a filariform or strongyloid character like that of the parasitic grand- 
parent. These gain access to the intestine of a new host in an unknown manner 
or shortly die. 

These worms may be found throughout the upper gastrointestinal tract, 
especially in the duodenum and upper part of the jejunum. The time elapsing 
between infections with the filariform larvae of the sexual generation and the 
appearance of the rhabditiform embryos of the parasitic type in the stools 
is between two and three weeks. The parthogenetic female types are usually 
called the strongyloides intestinalis, while the free sexual form is styled strongy- 
loides stercoralis. 

This form is found widely distributed in Indo-China, the East Indies, 
Africa, Europe, and North and South America. The mode of transmission to 
the second host is probably through the means of unfiltered water or of unclean, 
uncooked vegetables. 

(c). Trichotrachelidae. 
(1). Trichiuris trichiura. 

Synonyms. — Ascaris trichiura; trichocephalus trichiurus; trichocephalus 
hominis; trichocephalus dispar; trichocephalus mastigodes; whip-worm. 

The male worm is 35 to 45 mm. long; whitish; anterior three-fifths slender 
and thread-like; posterior two-fifths thicker, cylindrical, 
terminally rounded and curled; anus terminal; single spicule 
in a tubular sheath containing small spinules. 

The female parasite is 35 to 50 mm. long; shape as in 
male for front and body; posterior extremity straight, bluntly 
pointed terminally; vulva at beginning of thick posterior 
portion of body. 

The ova are very characteristic, brown, oval, thick- Fig. 44. — Tri- 
walled, with a colorless shining button-like protuberance at ^^1 ^e-" 1 a' 
each pole. The eggs are 50 to 54 microns long and 23 Male; B, female, 
microns wide, with an unsegmented yolk. Occasionally these ' yson ' 
eggs show variations in shades of brown, some being very much lighter 
than others. 

(2). Trichinella spiralis (trichina spiralis). 

The male worm is 1.4 to 1.6 mm. long and 0.04 mm. thick; cylindrical 
in shape; anterior end tapering, posterior end gradually and slightly thickening 
and terminating in a bifid extremity with two lateral somewhat conical tail 
appendages; cloacal aperture between these, which form a sort of bursa; back 
of cloacal aperture two pairs of papillae. 

The female worm is 3 to 4 mm. long; anterior end as in the male; posterior 
end nearly of same thickness to tail, which is rounded; anus terminal; vulva 
at anterior fifth of body; viviparous. 

The larvae when born are 90 to 100 microns in length, obtuse anteriorly, 
posteriorly prolonged to a pointed tail; when encysted as "muscle trichinae" 
the larvae measure about 1 mm. long and 0.04 mm. in thickness, tapering 




i5° 



DIAGNOSTIC METHODS. 



anteriorly, more thick and obtuse posteriorly with complete organization as in 
the adult and showing the characters of the different sexes. 

This parasite or its larvae are rarely if ever found in the stools. In its adult 
sexual stage it infests the intestinal tract of man and a number of mammalians, 




Fig. 45. — Trichinella spiralis: a, Gravid female, "intestinal trichiura"; E, embryos; 
G, vulva; Ov, ovary; b, adult male, "intestinal trichiura"; T, testicles; C, young larva; 
d, larva in musculature; e, encapsulated larva in muscle. (Tyson after Braun.) 



gives origin to a large number of larval worms, after which the adults die. The 
larvae pass through the muscular wall of the intestine and are carried by the 



THE FECES. 



151 



blood-current into various muscles of the host. Here they pass an indefinite 
encysted stage, a capsule forming around them and becoming calcified. l 

Herri ck and Janeway, 2 Packard, 3 and Cross 4 found the embryo in the blood, 
the latter in blood from an ear puncture. The blood was laked with 3 per cent, 
acetic acid, was centrifuged, and the sediment examined as outlined by Staubli. 5 
The chief means of infection of man with the trichinella spiralis is through 
the eating of insufficiently cooked pork, especially ham. 

(d). Strongylidae. 
(1). Uncinaria duodenalis. 

Synonyms. — Ancylostoma duodenale; strongylus quadridentatus; dochmius 
ancylostomum; sclero stoma duodenale; strongylus duodenalis; dochmius duo- 
denalis, and European hook-worm. 

The male parasite is whitish or blotched posteriorly with brownish when 
the intestine contains blood; 8 to 10 mm. long; cuticle finely striated transversely; 





Fig. 46. — Tail, with expanded bursa, of 
male uncinaria duodenalis. {Tyson.) 



Fig. 47. — Anterior 
end, showing mouth 
parts, of uncinaria 
duodenalis, dorsal 
view. (Tyson.) 



tapering to a blunt point anteriorly and with head curved upon the dorsum so 
as to give a slightly hooked anterior end; on each side of the median line on the 
ventral side of oral border two hook-like chitinous teeth and on dorsal border 
on each side of the median line one less curved chitinous tooth; with a dorsal 
conical tooth extending along back of oral cavity from the base of the cavity; 
in the oral cavity about the esophageal opening a delicate armature consisting 
of two dorsal and two ventral lancet-like pieces; posteriorly the body ends in 
an abruptly pointed tail in a copulatory bursal expansion of the cuticle, this 
having one dorsal and two lateral lobes; in the folds of the bursa one dorsal 
subdivided muscular ray, each division ending tridigitally, and on each side 
symmetrically placed an undivided dorsolateral, a divided lateral, undivided 
lateroventral, subdivided ventral and undivided small subventral muscular 
rays; cloacal aperture superterminal; two equal spicules. 

1 See Flury, Arch. f. exper. Path. u. Pharmakol., 1913, LXIIL 164; Gruber, Munch, med. 
Wchnschr., 1914, LXI, 645; Van Cott and Lintz, Jour. Am. Med. Assn., 1914, LXII, 680. 

2 Arch. Int. Med., 1909, III, 263. 

3 Jour. Am. Med. Assn., 1910, LIV, 1297. 

4 Arch. Int. Med., 1910, VI, 301. 

5 Kolle and Wassermann's Handbuch der pathog. Mikroorg., 1913, VIII, 73. 



152 DIAGNOSTIC METHODS. 

The female worm has the general appearance of the male and is shaped 
anteriorly like it; 12 to 18 mm. long; posteriorly tapering to a finely pointed 
tail; anus subterminal; vulva about the posterior third of the body length; 
two uterine and ovarian tubes. 

The ova are colorless, elliptical, thin-shelled with unsegmented or early 
segmenting material, 50 to 60 microns long and 30 microns broad. 

This worm has a wide distribution both in tropical and subtropical 
countries. Its habitat is in the duodenum, jejunum, and upper part of the 
human ileum. Infection with this organism is known as uncinariasis or 
ancylostomiasis. 1 A particularly severe type of anemia is set up by this para- 
site both through the influence of loss of blood and the elaboration of toxic 
hemolytic material by the parasite. The eggs of this organism are frequently 
found in the stools and should be carefully searched for in every case of severe 
anemia. The mode of infection by these organisms may be direct ingestion 
of dirty water or unclean vegetables, but it is probable that the most frequent 
method of infection is through the skin, these larvae attaching themselves to 
the feet of people walking in infested sand or water. The larvae penetrate 
the skin and make their way through the blood- and lymph-currents to the lungs, 
whence they penetrate to the air-passages and are supposed to be carried 
upward toward the mouth by the bronchial mucus and are then swallowed. 

(2). Uncinaria americana 2 (American Hook-worm) . 

The male parasite differs from the former organisms discussed in being 
of smaller size (6 to 9 mm. long and more slender than the duodenalis), in the 
smaller size and more conical shape of the head, in having no hooklets on the 
oral rim, but instead on each side a large ventral and smaller dorsal chitinous lip, 
extending from the the rim toward the median line; in a greater prominence and 
projection into the oral cavity of the dorsal conical tooth; in the smaller size of 
the copulatory bursa, its dorsal lobe being subdivided and the ventral margin 
being extended so as to form an indefinite ventral lobe; and showing the dorsal 
muscular ray of the bursa divided, each division ending in a bipartite tip. 

The female worm differs from the duodenalis in being shorter and more 
slender (8 to 15 mm. long), with similar differences of the anterior end as above 
outlined for the male; vulva just in front of the middle of body length instead 
of at the posterior curve as in the uncinaria duodenalis. 

The ova of this parasite are somewhat larger (68 to 70 microns long and 
38 to 40 microns broad) than those of the uncinaria duodenalis, but are other- 
wise similar. 

Stiles has found that these parasites 3 are the common cause of the frequent 
"anemia of the South." Smith believes that uncinariasis exists in every case 
in which " ground-itch" has occurred within eight years and that the disease 
is rarely ever present in those who have not had this condition during that 

1 See Bruns, Kolle and Wassermann's Handbuch, 1913, VIII, 41. 

2 Also called Necator americanus. See Stitt, Jour. Am. Med. Assn., 1912, LIX, 1706; also, 
Stiles, Pub. Health Reports, 1913, XXVIII, 7; also, Ashford, War Dept., Office of Surgeon 
Genera], Bull. 2, 1913, 59 and 72; Ferrell, Jour. Am. Med. Assn., 1914, LXII, 1937. 

3 See Glover, Jour. Am. Med. Assn., 1912, LVIII, 1837, for a discussion of uncinariasis 
in oriental immigrants. 



THE FECES 



*53 



period. This would point to the general transmission of infection through 
the skin. The uncinaria duodenalis has long been known as the cause of the 
so-called Egyptian chlorosis, tunnel- workers' anemia, brick-layers' anemia, 
and other conditions necessitating work in low-lying watery places. 

Pseudoparasites. 

It not infrequently happens that extraneous substances are found in the 
feces which very closely simulate a parasite or its ova in appearance. These 
substances are, for the most part, food residues and should be carefully differen- 
tiated by applying tests for cellulose, which will show in all vegetable cells. 

Stiebel has described, under the name of diacanthos polycephalus, a frag- 
ment of the woody portion of a bunch of raisins. Sultzer describes a mulberry 
seed as a vesicular worm under the name of ditrachyceros rudis. Bastiani 





Fig. 48. — Tail, with expanded 
bursa, of male uncinaria ameri- 
cana. {Tyson.) 



Fig. 49 — Anterior end 
of uncinaria americana, 
showing mouth parts 
(dorsal view). {Tyson.) 



considers the larynx of a bird which he found in the fecal material as being 
a biped worm, under the name of sagiiula hominis. Scopoli regards a frag- 
ment of the trachea of a bird as an entozoon form under the name of physis 
intestinalis. A pupil of Moquin-Tandon describes a strip of lettuce under 
the name of striatula, regarding it as a worm intermediate between the ascaris 
and the oxyuris (Guiart and Grimbert). 1 

Perhaps the most frequent pseudoparasite of the feces is the pulp of an 
orange, it shows in the feces in the form of large oblong masses terminated 
by two slender extremities, one of which ends in a sort of parenchyma. These 
vesicular masses are the large cells which secrete the orange juice and which 
are generally found intact in the fecal material. These have been frequently 
mistaken for hydatids or for parasitic ova. Certain spores, such as those 
of the truffle or of lycopodium, have not infrequently been mistaken for para- 
sitic eggs. Moreover, one may find in the fecal material the pollen of the 
coniferous plants which very closely simulates parasitic ova. The spines which 
form the down on certain fruits, such as raspberries, strawberries, peaches, 
and quinces, so closely resemble parasites that careful study is in some cases 
essential. 

1 Franca viglia (Policlinico, 1915, XXII, 1052), reports the presence of blood clots simu- 
lating parasites. 



154 



DIAGNOSTIC METHODS. 




IO.M 20. 30. 40. SO. 60. 70.y 80. SO. 



I20- I30. i*0. ISO. 160. I70. ISO. 190. 2C0. 



Fig. 50. — Parasitic bodies, ova, and 
{Tyson.) 

1. Larval'strongyloides intestinalis. 

2. Ovum of fasciola hepatica. 
5. Ovum of taenia nana. 

4. Ovum of uncinaria duodenalis. 

5. Ovum of uncinaria americana. 

6. Ovum of taenia saginata. 

7. Ovum of taenia solium. 

8. Ovum of opisthorchis sinensis. 



larvae met in human feces; color approximate only. 

9. Ovum of opisthorchis felineus. 

10. Ovum of cotylogonimus heterophyes. 

11. Ovum of taenia cucumerina. 

12. Ovum of ascaris lumbricoides. 

13. Ovum of dicroccelium lanceatum. 

14. Ovum of bothriocephalus latus. 

15. Ovum of trichiuris trichiura. 

16. Ovum of oxyuris vermicularis. 



PLATE VIII. 





!% 



i 7 .< 









?rf - 










K^aTharme H\l\ 



Vegetable Cells found in Feces. (After Schmidt and Strasburger.) 



THE FECES 1 55 

It is, therefore, wise in all cases before pronouncing a finding as one of 
a parasite to be perfectly sure of your ground. 

BIBLIOGRAPHY. 

i. Braun. Die tierischen Parasiten des Menschen. Wurzburg, 1908. 

2. Briancon. De L'Ankylostomiase. Lyon, 1904. 

3. Cammidge. Feces of Children and Adults. New York, 19 14. 

4. Craig. The Parasitic Amebas of Man. Philadelphia, 19 n. 

5. Hecht. Die Faeces des Sauglings und des Kindes. Wien, 1913. 

6. Hemmeter. Diseases of the Intestines. Philadelphia, 1902. 

7. Lynch. Coprologia. Buenos Ayres, 1896. 

8. Mtjsgrave and Clegg. Amebas: Their Cultivation and Etiologic Significance. 
Manila, 1904. 

9. Nothnagel. Beitrage zur Physiologie und Pathologie des Darmes. Wien, 1895, 
Krankheiten des Darmes. Wien, 1900. 

10. Von Oefele. Technik der chemische Untersuchung des menschlichen Kothes, 
Leipzig, 1908. 

11. Schmidt und Strasburger. Die Faeces des Menschen. Berlin, 191 5. 

12. Van Ledden Hulsebosch. Makro- und mikroskopische Diagnostik der mensch- 
lichen Exkremente. Berlin, 1800. 



CHAPTER V. 

PARASITES. 

I. General Considerations. 

In the previous portions of the work, the writer has introduced those 
parasites which are more particularly related to the parts under discussion. 
There are, however, a large number of organisms which do not fall naturally 
within the scope of any of the chapters outlined for this book. These will, 
therefore, be discussed in general without much regard to distinct classifications, 
as many of the subdivisions have been treated previously. This discussion is 
taken, for the most part, from Tyson. * 

II. Trematodes (Fluke- worms). 

Flukes are naked and unsegmented flat worms, usually of the shape of a 
leaf or of the tongue (occasionally pyramidal or elongated and more or less 
cylindrical), provided with incomplete digestive canal (without anus), possess- 
ing one or more suckers and occasionally hooklets; with but few exceptions 
hermaphroditic and, as a rule, presenting a complicated series of metamorphoses 
in their development. 

In structure it is customary to speak of the surface upon which the genital 
pore opens as the ventral; this surface commonly shows also the orifices of 
the mouth and one or more suckers. On the dorsal surface in many occurs the 
opening of a small canal, spoken of as Laurer's canal, of unknown function. 
The surface of the body is covered by a fairly thick and firm cuticle, often 
provided over variable areas with small spines or tubercles. Beneath the 
cuticle over its internal surface is spread the superficial muscular layer (not 
showing the structure of muscle of higher animals, however), with longitudinal, 
circular, and diagonal fibers, while within this along the borders is met the 
parenchymatous muscle. The general internal tissue of the body, spoken of 
as the parenchyma, is a fine reticular connective tissue which closely surrounds 
the various organs. 

The suckers of trematodes vary in number and arrangement on the anterior 
and posterior extremities, the ventral surface and its borders, and in a few 
cases also on the dorsal surface. Usually the oral opening is surrounded by 
such a sucker and in addition, in the forms likely to be met with in man, on the 
ventral surface some distance posterior to the oral sucker is a second, known 
as the ventral sucker or acetabulum, in the median line. Not infrequently in 
the lining of these suckers, on their lips, or on the cuticle close to the lips, 
chitinous hooklets are to be found. 

1 See Ward, Illinois Med. Jour., 1912, XXII, 417; Cort, 111. Biological Monographs, 1915, 
I, No. 4. 

156 



PARASITES. 



157 



The alimentary system consists of a mouth, opening in the oral sucker and 
situated terminally or on the ventral surface of the anterior end of the worm. 
This cavity continues into a dilated tube with thick walls, the pharynx, this 
extending posteriorly by a short, straight, and usually narrow esophagus, which 
divides in the anterior portion of the body into the two intestinal tubes or ceca. 
At the posterior extremity of the body is a small orifice, the excretory pore, 
which serves as outlet for a series of more or 
less complex canals for the convection of the 
fluid waste, the arrangement representing a 
low nephridial apparatus, while the mouth 
serves as an anus. The reproductive system 
is highly developed, showing numerous 
minor variations in the different genera 
and species. 

Adult flukes are parasitic upon a wide 
range of animal life, including the higher 
animals, fish, amphibia, reptiles, and birds, 
living as ectoparasites and endoparasites. 
The varieties affecting man are compara- 
tively few. The most common parts of the 
human body to be infested are the intes- 
tines, gall-ducts, respiratory tubes, and 
blood-vessels. 



(a). Fasciolidae. 
(1). Fasciola hepatica. 

Synonyms. — Distomum hepaticum; dis- 
tomum cavice; fasciola humana, cladoccelium 
hepaticum; common liver-fluke. 

A comparatively large fluke, measuring 
20 to 50 mm. long and 8 to 13 mm. wide, of 
leaf shape, with anterior extremity pro- 
longed into a small cone; greatest width of 
body about the anterior third of length; 
light brown color; cuticle provided with 
alternating transverse rows of spines, ex- 
tending on ventral surface to the posterior 

level of testes, but not as far posteriorly on the dorsal surface; the oral 
sucker at the anterior end of cephalic cone, inclining to ventral surface, 
1 mm. in diameter; ventral sucker near anterior end behind cephalic cone, 
1.6 mm. in diameter; well developed larynx and short esophagus; intestinal 
branches extending nearly to the posterior extremity of the worm, ap- 
proaching the median line posteriorly with few median and numerous 
lateral branches; excretory pore at posterior extremity, with well-developed 
system of excretory tubes; genital pore in the median line anterior to the ventral 
sucker; two large highly branched testes, mostly posterior to the transverse 




Fig. 51. — Showing the sexual 1 glands 
of fasciola hepatica; 5X1. O, oral 
sucker; D, intestinal ceca; Do, vitelline 
glands; Dr, ovary; Ov, uterine canal; 
T, testicles; Sq, "shell gland;" V, trans- 
verse vitelline duct; Gp, genital pore; 
S, ventral sucker. ( Tyson after Braun.) 



158 



DIAGNOSTIC METHODS. 




vitelline duct; ovary single, branched, lying in front of testes and to one side 
of the median line; the uterus coiled into a rosette, showing as a brown spot 
just back of the ventral sucker on the ventral surface; vitelline glands numerous, 

ranging along each lateral border from the 
level of the ventral sucker to the posterior 
extremity of the worm; vitelline ducts run- 
ning transversely at about the end of the 
anterior third of the body. The ova are 
yellowish-brown, oval, operculated, and 
measure 130 to 145 microns in length and 
70 to 90 microns in width. 

This fluke is a common one in most 
mammalians and has a wide geographical 
distribution over the world, being found not 
infrequently in America. Its usual habitat 
is the gaU-ducts, but it has been seen in the 
gall-bladder, intestines, in the portal and 
other venous channels and in subcutaneous 
cysts. The ova appear in the feces and are 
the chief means of diagnosis of this condi- 
tion. The embryo which develops from 
the ovum completes its developmental cycle 
in the body of a snail, the Limnaea truncatula. 
From the body of the snail the cercariae 
escape in the water and become attached to 
grass or other aquatic material, which is 
taken in by the animal. Infection in man 
is quite rare, about 32 cases having been 
reported in the literature. 




Fig. 52.— Fasciolopsisbuski: a, oral (2). Fasciolopsis buski. 

r^m-gtndtT; &S33rj SY*oxvxs.-Dis,o,nu m buski; dislomum 

/and g, posterior and anterior testicles ; eras sum. 

h, ovarv; i, cecum; k, uterus. (Tyson m, 1 ,1 £ ,»• • • • 11 

after Braun.) Tne length of this organism is variable, 

ranging between 24 and 70 mm.; breadth 
5.5 to 14 mm.; lance-shaped; narrowing more rapidly anteriorly than poste- 
riorly, maximal width about the middle of the length; no cephalic cones; 
brownish in color; cuticle without spines; oral sucker small and placed on 
ventral surface of anterior extremity; ventral sucker two or three times as large 
as the oral, placed near anterior end and showing a saccular distention extend- 
ing posteriorly; very short esophagus, back of the fairly developed pharynx; 
the two ceca without branches; genital pore at the anterior quarter of the 
acetabulum; cirrus pouch large; testicles branched; in posterior part of body 
one back of the other; uterus in anterior half of body, tortuously coiled; ovary 
at middle of length of body, to the right of median line; Laurer's canal present; 
vitelline follicles numerous along lateral margin from the level of the ventral 



PARASITES. 



J 59 



sucker to the posterior extremity; the transverse vitelline ducts at the equator 
of body. The ova are brownish, ovoid, operculated, and measure 125 microns 
in length and 75 in width. 

Very little is known of the intermediate stage and of the host of Busk's intes- 
tinal fluke. This has been found in the small intestine 
of man and probably arises from eating infected food. 

(3). Opisthorchis felineus. 

Synonyms. — Distomum conus; distomum lanceolatum 
(Siebold) ; distomum sibiricum; distomum tenuicolle; the 
European cat-fluke. 

Variable in size according to the state of contrac- 
tion, 8 to 11 mm. long and 1.5 to 2 mm. broad; yellow- 
ish-red and nearly transparent; flat and lanceolate; 
anterior end constricted and attenuated into a cone; 
posterior end more obtuse; cuticle without spines; oral 
sucker toward the ventral surface at anterior extremity; 
ventral sucker at base of cone about one-fourth of 
body length; pharynx and esophagus of equal length; 
ceca comparatively straight and unbranched, reaching 
nearly to the posterior extremity and often seen filled 
with blood; excretory pore terminal; its tubular vesicle 
winding in the median line between the testes and 
branching in front of the anterior testes; testes in the 
posterior part of body, the anterior four-lobed, the other 
five-lobed; cirrus and pouch absent; genital pore in the 
median line in front of the ventral sucker; slightly lobate 
ovary in the median line anterior to the testes; recepta- 
culum seminis prominent; uterus anterior to the ovary 
and testes coiled in the middle third of the body; vitelline 

follicles occupy about the middle third of the body, beginning anteriorly at 
the level of the ventral sucker. The ova are oval in shape, operculated, and 
measure 30 microns in length and 11 in width. 

This worm has been found in the gall-ducts of man and other animals, 
especially in Russia, Siberia, Hungary, and Japan. 

(4). Opisthorchis sinensis. 

Synonyms. — Distomum sinense; distomum spathulatum; distomum hepatis 
endemicum seu perniciosum; distomum hepatis innocuum; distomum japonicum; 
the Japanese or Chinese liver-fluke. 

This parasite measures 10 to 20 mm. in length and 2 to 5 mm. in breadth; 
long and lanceolate; reddish and nearly transparent when fresh; cuticle with- 
out spines; oral larger than ventral sucker, on the ventral face of anterior 
extremity; ventral sucker about one-fourth of body length posterior to former; 
pharynx and esophagus small and a bifurcation of the latter close to oral 
sucker; ceca unbranched, reaching close to the posterior extremity; excretory 
system as in the previous species; in the posterior fourth of body the two 




Fig. 53 . — Opisthor 
chis felineus; from liver 
of cat; 10 x 1. {Tyson 
after Braun.) 



i6o 



DIAGNOSTIC METHODS. 



testes, one in front of the other, with from four to six dendritic branches; 
no cirrus or pouch; genital pore in the median line just in front of the 
acetabulum; ovary tri-lobed and placed just anterior to a large gourd-shaped 
receptaculum seminis both in the median line and anterior to testes; uterus 
well-developed and coiled in the middle area of the body between the ovary 
and ventral sucker; vitelline follicles in the marginal fields along the middle 
third of the body. The ova are brown, oval, operculated, and measure 27 

to 30 microns in length and 15 to 17 in width. 

This fluke is comparatively common in Japan 
and eastern Asia and has been reported in America. 
It infests the gall-ducts and has been found both 
in the pancreatic duct and intestine. Little is 
known of the intermediate hosts. 

Many other types of these distomata are met 
with in the liver of various animals and are occa- 
sionally found in man. Among these we find the 
distomum lanceolatum, distomum heterophyes, 
distomum conjunctum, and amphistomum hominis 
(gastrodiscus hominis). 

III. Nematodes (Round Worms). 

Eustrongylus gigas. 

Synonyms. — Ascaris canis et martis; ascaris 
visceralis et renalis; strongylus gigas; strongylus 
renalis; eustrongylus visceralis. 

The male worm is red in color; 14 to 40 cm. 
in length and 4 to 6 mm. in thickness; slightly 
tapering anteriorly; mouth terminal; with a hex- 
agonal orifice surrounded by six lips bearing 
papillae; cuticle thin and transparent, finely 
striated transversely; about 150 papillae along 
the longitudinal lines laterally; caudal extremity 
with an oval plate-like expansion serving as a 
bursa, its margin bearing small papillae and 
slightly indented dorsally and ventrally; single 
sexual spicule. 

The female parasite shows the general ap- 




Fig. 54. — Opisthorchis sin- 
ensis: ventral surface, stretched; 
a, oral sucker; b, ceca; c, geni- 
talpore; d, acetabulum; 
e, uterus; /, vitelline glands; 
g, ovary; h, receptaculum sem- 
inis; /, Laurer's canal; i, test- 
icles; k, excretory canal; m, ex- 
c r e t o r y pore. {Tyson after 
Braun.) 



pearance and head end as in the male; 20 to 100 
cm. in length and 5 to 12 mm. in thickness; caudal extremity obtuse, straight, 
with anus subterminal; vulva 50 to 70 mm. posterior to mouth; single 
ovarian and uterine tube plicated from near the anterior end along the in- 
testine nearly to the anus, then returning to the vulva near the anterior 
end. 

The ova are brown, ellipsoid, with thick shell marked by external cribri- 
form depressions, 64 to 68 microns in length and 40 to 44 microns in breadth. 



PARASITES. 



161 



This worm, which is more common in the dog, has been reported a num- 
ber of times in man. It is the largest of the nematode worms and has its 
habitat in the pelvis of the kidney. Little is known of its life history. 



IV. Parasites of the Skin. 

Arthropoda. 

These are bilaterally symmetrical segmented animals whose segments 
do not correspond, but vary in structure, and which primitively bear upon each 
segment a pair of jointed appendages. The segments are often more or less 
fused, thus forming special body-regions which may themselves be more or less 




Fig. 55. — Eustrongylus gigas: female, natural size, in kidney of dog. 
(Tyson after Railliet.) 

fused together as well. The covering of these animals is a comparatively 
thick and strong cuticle which remains pliable between the segments of the 
body and of the jointed appendages, but which commonly becomes hard and 
shell-like from chitinous or calcareous material directly over the different body- 
segments and internodes of the jointed appendages. This arrangement re- 
quires that in the growth of the individual the firm external covering should 
from time to time be shed, such changes taking place periodically and being 
known as moults. While each segment in the primitive animal is provided with 
a pair of jointed appendages, these in the individual species are often lost 
from this or that part of the body, or may remain rudimentary and inconspicu- 
ous or may take on special features of structure from the assumption of special 
function which causes their wide departure from the original and common type 



l62 



DIAGNOSTIC METHODS. 



used for locomotion. The arthropods commonly reproduce by ovulation, 
the development of the embryo to the adult often showing more or less compli- 
cated metamorphoses. The true parasitic forms of the arthropoda thus far 
met in man are limited to the Arachnoids and Insects. 

(A). Arachnoidea. 
(a). Sarcoptes or acarus scabiei (the Itch Parasite). 

This parasite is oval in shape, is provided with horns and bristles, is barely 
visible to the naked eye, the male being from 0.2 to 0.3 mm. in length by 0.145 
to 0.19 mm. in breadth; the female is somewhat larger, showing a length of 0.33 
to 0.45 mm. and a breadth of 0.25 to 0.35 mm. 

The female lies at the end of a burrow in the epidermis, in situations 
where the skin is most delicate, as between the fingers, at the elbows, under 





Fig. 56 — Acarus scabiei: A, female, dorsal view; B, portion of human epidermis, show- 
ing burrows with contained ova and young acarians. (Gould.) 



the knees, and in the groin. In this burrow, which varies from a few milli- 
meters to a centimeter in length, the female deposits her eggs, after which she 
dies. The eggs hatch in from 4 to 8 days, and in about 14 days the larvae 
are sufficiently matured to make their own burrows. 

The disease is communicated either by the clothing or by personal contact. 
To demonstrate the parasite, the burrow is opened with a needle and the female 
pressed out on a slide, which is then covered and examined. 

(b). Demodex folliculorum. 

This parasite is very small, varying in length from 0.3 to 0.4 mm. It 
is somewhat cylindrical, tapering to an obtuse point at the posterior end. 
This parasite has its habitat in the sebaceous follicles, especially of the face and 
nose. 

(c). Leptus autumnalis (Harvest-bug). 

This parasite is also known as trombidium irritans or red jigger. This 
is a minute red parasite, from 0.3 to 0.5 mm. long, .which has three pairs of 
legs, with rows of bristles upon its back and belly. It prevails in summer on 



PARASITES. 



163 



grass and plants and attaches itself to the skin of man by its hooklets. The 
condition of the skin due to the infection with this parasite is known as trom- 
bidiosis. x 



(B). 
(a 



Insecta. 
). Hemiptera. 
(1). Pediculus capitis (Head-louse). 

The male is from 1 to 1.5 mm. long, the female 1.8 
to 2 mm. long. The color of the parasite varies some- 
what with the race of its host. In the Caucasian it is 
gray with a dark border, in the Negro and Chinamen it is 
much darker in color. The 
eggs are 0.6 mm. in length 
and are attached to the hairs, 
forming the so-called "nits." 
These nits are whitish oval 
masses which are easily visible. 

This parasite, while usually 
found upon the hair of the head, 
may be found in other portions 
of the body. 2 The symptoms 
may be severe or very slight. 





Fig. 57. 
Demodex folli- 
culorum: from 
dog, enlarged. 
(Tyson after 
Braun.) 



vesti- 



Fig. 58. — Leptus autum- 
nalis: enlarged. (Tyson after 
Braun.) 



(2). Pediculus 
menti (Body-louse). 

This parasite is consider 
ably larger than the former, being from 2 to 5 mm. long 
and whitish-gray in color, the back part of the body being 
wider than the thorax. The antennae are longer than are those of the head- 
louse. The eggs are from 0.7 to 0.9 mm. in length, about 70 being laid by 
each female. 

This parasite is found upon the clothing in which it deposits its eggs, 
especially about the neck, back and abdomen. 

(3). Pediculus pubis (phthirius inguinalis or Crab-louse). 

This parasite is smaller than the head-louse, grayish-yellow or grayish- 
white in color, the male being from 0.8 to 1 mm. in length, the female about 
1. 1 2 mm. in length. The eggs are pear-shaped, from 0.8 to 0.9 mm. in length 
and from 0.4 to 0.5 mm. in breadth. 

This parasite infests the parts of the body covered by the shorter hairs, 
such as the pubis, axilla, eye-brows, and chest. 

1 See Olson, Jour. Am. Med. Assn., 191 5, LXIV, 2060. 

2 Goldberger and Anderson (Pub. Health Rep., 1912, XXVII, 297) show that typhus fever 
may be transmitted by this parasite as, also, by the body louse. See, also, Nicolle, Comte and 
Conseil, C. R. Acad. Sc, 1909, CXLIX, 486; Ricketts and Wilder, Jour. Am. Med. Assn., 
1910, LIV, 1304; Goldberger and Anderson, Pub. Health Rep., 1910, XXV, 177; Ibid., 1912, 
XXVII, 835; and Jour. Am. Med. Assn., 1912, LIX, 514. 



164 



DIAGNOSTIC METHODS. 



(4). Cimex lectularius (acanthia lectularia or Bed-bug). 

While, strictly speaking, the bed-bug is not a parasite of man, yet as its 
habitat is the bed, bedding, and walls of the sleeping apartment of man, it 
may be considered as indirectly parasitic. It usually emerges at night from 




v^> 




Fig. 59. — Pediculus capitis: X 
{Tyson after Braun.) 



Fig. 60. — Pediculus vesti- 
menti: xio. {Tyson after 
Braun.) 



its lodging for the purpose of securing its nourishment in the blood of its victims. 

This parasite is reddish-brown in color, oval in shape, from 4 to 5 mm. 

in length and 3 mm. in breadth. These insects, if crushed between slides 

or as more usual between the hand and a part of the victim's body, have a 




Fig. 61. — Pediculus pubis. {Tyson after Braun.) 



characteristic odor very much resembling kerosene. The blood is drawn from 
the victim by means of a long proboscis. The eggs are approximately 1.12 
mm. in length and require about 11 months for their development to the 
sexually ripe insect. These eggs are retained in the crevices of the bed, floors, 



PARASITES. 165 

furniture, wall-paper, and other parts of the dwelling so that the complete 
removal of these eggs and parasites is a matter of some difficulty. 1 

That these insects have more or less importance from the standpoint 
of transmission of disease from one person to another must be remembered. 
Individuals vary in their susceptibility to the bite of the bed-bug, some being 
indifferent to it while others are markedly affected by it. 




1 



Fig. 62. — Pulex irritans: X 14 {Tyson after Braun.) 

(b). Diptera. 
(1). Pulex irritans (Common Flea) . 

The male is from 2 to 2.5 mm. in length, the female as much as 4 mm. 
It is a red or brownish-red insect, having a laterally compressed body, an oral 
haustellum, serrated soft mandibles, a tongue sheathed in an inferior labium, 
and a pair of labial four-jointed palpi. Each of the triple segments of the 
thorax bears a pair of five-jointed double-clawed legs. The female deposits 
her eggs, not on the human being, fortunately, but in the fissures, crevices, 
or holes of garments or furniture which may be accessible. 

(2). Pulex penetrans (Sand-flea or Jigger). 

This parasite is a minute, brownish-red, egg-shaped insect which penetrates 
the skin of man. The female is the infecting insect and produces painful 
irritation and even suppuration. 

Vegetable Parasites. 

(1). Achorion Schbnleinii. 

This organism is the cause of the disease known as favus or tinea favosa. 
This fungus invades the root sheaths, the bulbs, and the shafts of the hair 
filaments of the scalp, but it also occurs upon the "non-hairy" portions of 
the skin and upon the nails. 2 The spores gain access to the deeper layers of 
the skin and develop around the hair-shaft, forming a characteristic yellowish 
cup-shaped crust which has a peculiar mouse-like odor. 

1 See Rucker, Pub. Health Reports, 1912, XXVII, 1S54; Thomson, Ann. Trop. Med. and 
Parasitol., 19 14, VHI, 19. 

2 See Foster, Jour. Am. Med. Assn., 1914, LXIII, 640. 



i66 



DIAGNOSTIC METHODS. 



In searching for this parasite, a favus crust is softened by the addition 
of a few drops of water or dilute sodium hydrate solution and placed upon 
a slide and examined with the high-power dry lens. The hairs may also 
be examined in the same manner or may be stained by methods outlined in the 
discussion on Tinea tricophytina. 

The mycelial threads appear as narrow, flattened, ramifying, short or 
elongated, linear cells or tubes, which may be simple and empty, or be divided 
more or less regularly by transverse partition walls transforming the longer 
and simple into shorter and compound cells. The latter often contain in their 
cavities sporules clinging to either side, in which case the mycelial threads 
are termed sporophores. The conidia are encapsulated or are strung together 




Fig. 63. — Pulex penetrans: young female, enlarged. (Tyson after Braun.) 



like the beads of a necklace, and appear as round, oval, angular, or very irregu- 
larly contoured bodies. These mycelial threads branch at right angles, the 
spores measure from 3 to 10 microns in diameter (Hyde). 

(2). Trichophyton megalosporon endothrix. 

This organism is the cause of tinea circinata (herpes tonsurans, ring- 
worm of the body), and of tinea sycosis (hyphogenous sycosis, tinea barbae, 
ringworm of the beard, barber's itch). 

The trichophyton is composed of spores which vary greatly in size, but 
which, as a rule, are somewhat larger than those of the type next to be discussed. 1 
They are frequently cuboidal, oval, or irregularly rounded, but their chief 
characteristic lies in their arrangement in lines or chains, extending up and 
down the hair shaft. The mycelium is found without, but never within the 
hairs (Hyde). 

These fungi may be stained by the method of Morris and Calhoun. The 
hair is first washed in ether to remove all fatty debris; it is then put for one 

1 Priestley (Ann. Trop. Med. and Parasitol., 1914, Mil, 113) reports the presence of the 
microsporon scorteum in a case of ringworm. 



PARASITES. 



167 



or two minutes in Gram's iodin solution and is stained after drying for from 
one to five minutes in gentian-violet. It is again dried and treated for a minute 
or two with the iodin solution and for an equal length of time in aniline oil 
containing pure iodin, after which it is cleared with aniline oil, washed in xylol, 
and mounted in Canada balsam. 

(3). Microsporon audouini (Trichophyton Microsporon). 

This parasite appears under the microscope chiefly in the form of a large 
number of round spores, irregularly grouped or massed about the follicular 
portions of the hair. Mycelial threads, large and branching, are often seen 




Fig. 64. — Achorion schonleinii, X 500 diameters. (Van Harlingen.) 

within the hair. The sheath of spores surrounding the hair is often con- 
tinued upward for 1/16 to 1/8 inch above its exit from the follicle and may 
be recognized as a whitish or grayish coating of the hair. These mycelial 
threads are all within the hair proper, thus differing from those of the tricho- 
phyton which are never within the hair; after repeatedly dividing and subdivid- 
ing they terminate on the outer surface of the shaft in fine filaments, at the 
extremities of which are the spores. This parasite is the cause of the disease 
tinea tonsurans, or ringworm of the scalp. 

(4). Microsporon furfur. 
This parasite is readily recognized by the microscopic examination of the 
scales scraped from the skin. Innumerable clustered spores, highly refractive 
and resembling in their circular and oval contours droplets of oil, are quite 
characteristic. The mycelial threads are not usually branched, but lie in a 
close network, among which sporophores are distinguishable, with conidia 



i68 



DIAGNOSTIC METHODS. 




mm 



W0 

mi 

mm 



WW 



Fig. 65. — Xormal hair, x 90c. 



mm 




H\H 



Fig. 66. — Hair showing trichophyton endo-ectothryx, X 900. 



PARASITES. 



169 



and terminal elements emerging at one extremity of the spore case. Both 
elements of this organism are more readily stained by the aniline dyes than are 
those of the trichophyton or favus. This organism is the cause of the con- 
dition known as tinea versicolor. 

(5). Microsporon minutissimum. 

This organism is the etiologic factor of erythrasma. It is characterized 
by the extreme delicacy and fineness of its threads and very minute spores. 
The threads are either simple, cylindrical bodies of variable size or they 
may exhibit partition septa, may divide dichotomously, and may terminate 
in hooked or knobbed expansions. The largest transverse diameter is 0.6 
micron; in length the mycelium presents the greatest variations. 




FlG. 67. — Hair showing microsporon audouini, X 900. 



(6). Blastomycetes. 

These organisms may be found in the cutaneous eruptions of the skin 

in blastomycosis and may be described as follows, according to Montgomery 

and Ormsby. In unstained preparation the organisms appear as round or oval 

bodies with a double-contoured highly refractive capsule. Within the capsule, 

in many instances, granules or spore-like bodies can be distinguished. The 

addition of a 1 to 10 per cent, solution of potassium hydrate to the specimen 

under examination facilitates the recognition of these bodies. In stained 

sections the double contoured, homogeneous capsule is usually separated from 

a finely or coarsely granular protoplasm by a clear space of varying width. 

Vacuoles of different sizes are found in some organisms. In both pus and tissue, 

organisms in pairs or in various stages of budding are commonly seen. 1 The 

1 See Whitman, Jour. Infect. Dis., 1913, XIII, 85. See a long series of articles by various 
authors in Arch. Int. Med., 1914, XIII, 509 ff. 



170 



DIAGNOSTIC METHODS. 



parasite, as a rule, varies in size from 7 to 20 microns, though slightly smaller 
and much larger forms occur in some cases. 

The organisms are readily obtained in pure culture from unbroken ab- 
scesses, from miliary abscesses in the borders of the cutaneous lesions, and from 
the miliary nodules and abscesses in the deep-seated organs. The peculiarities 
of the cultures of blastomycetes must be looked for in other works. Micro- 
scopically, the organism obtained in culture appears at first as a fine, branching 




Fig. 68. — Mycelial threads of blastomyces from old agar culture. 
(From photograph by W. A. Pusey.) 



mycelium with a few small spore-like bodies. Later a large, segmented, often 
pod-like mycelium appears, together with large, round, or oval bodies with 
bud-like projections. 

The condition known as coccidioidal granuloma, which is especially observed 
in California, has often been confused in the literature with blastomycosis. It 
is, however, now well established that the etiologic factor of this disease is the 
coccidioides immitis, an organism somewhat similar to but differing distinctly 



PARASITES. 171 

in many respects from the blastomyces. This organism was first reported by 
Posadas and Wernicke, but was later classified by Rixford and Gilchrist and 
cultivated by Ophiils. 1 

(7). Sporothrix Schenckii. 
This organism is the cause of sporotrichosis and was first recognized 
by Schenck, Hektoen and Perkins. 2 Since its discovery a large number of 
cases have been identified in all parts of the world, especially in the middle- 
western portions of the United States. The lesions in this condition 
resemble, at times, those of tuberculosis, while, occasionally, they appear 
more like those of syphilis. The chief characteristics are the sharply 
defined, painless, cutaneous or subcutaneous abscesses which follow the lym- 
phatics and do not yield to the ordinary surgical procedures. In living tissues 
the sporothrix organisms appear as elongated or oval bodies fairly uniform in 




Fig. 69. — Budding forms of Sporothrix Schenckii in pus, X 1200. Gram's Stain. (Cour- 
tesy of Dr. D. J. Davis.) 

size and often showing distinct budding processes. The arrangement is usually 
single, but frequently two or more are found end to end or arranged radially 
about a central spore. Branched mycelial filaments are not found in the living 
tissues but they develop abundantly on artificial media, forming, at their sides 
and extremities, numerous spores, oval in shape and differing decidedly in 
appearance from the typical tissue forms. These latter forms may be found, 
however, in small numbers in artificial cultures (Davis). Smears from the 
lesions are made in the usual way. To obtain cultures proceed as follows: 
Some of the light yellow pus is taken, by aseptic methods, from the suspected 
lesion, is transferred either to glucose or maltose agar, or blood agar, and is kept 
at room temperature. After a few days small points appear from which circular 
growths with marked striations develop in 10 to 12 days. Later these may show 
a deep black pigmentation. Smears made from these cultures show a typical 
sporothrix, the mycelium being coarse (2 fi broad) and forming a dense, branch- 

1 See MacNeal and Taylor, Jour. Med. Research, 1914, XXX, 261; Cooke, Arch. Int. 
Med., 1915, XV, 479; Brown and Cummings, Ibid., 608. 

2 See Gougerot, in Kolle and Wassermann's Handb. d. path. Mikroorg., 1912, V, 211. 



172 DIAGNOSTIC METHODS. 

ing, septate network. Spores (4 to 5 ^ long and 2 to 3 \x broad) develop by bud- 
ding either from lateral or terminal filaments or from the sides of the thread. 
The organism stains readily with the ordinary dyes and is, also, Gram-positive. ■ 

(8). Negri Bodies. 

Considerable discussion has centered about these bodies as the etiologic 
factor of rabies (hydrophobia), but now, thanks to the successful experiments 
of Noguchi 2 in cultivating them, the subject must be regarded as practically 
settled in the affirmative. These bodies are found in almost all of the nerve 
cells of the central nervous system but are more numerous and more easily 
found in the cells of Ammon's horn, cortical tissue in region of the fissure of 
Rolando, and in the Purkinje cells of the cerebellum. They are also present 
in the salivary glands of the infected animal but are not readily detected therein. 
Their demonstration in the cells of the brain is regarded as equivalent to a 
positive diagnosis of rabies. As they have never been found in any condition 
other than hydrophobia, they must be regarded as specific. 

These bodies were originally described by Negri, 3 although Galtier 4 and 
Pasteur 5 had previously demonstrated the infectious character of rabies. Since 
that time many workers have confirmed their findings and have added much to 
our knowledge of these bodies. 6 

They are found lying in the cytoplasm of the nerve cells or in their branches, 
occasionally outside, and appear as round, oval, triangular, pear-shaped, spindle- 
or sausage-shaped bodies. They vary in size from 0.5 n to 20 y. in diameter, the 
size increasing with the stage of the disease. They show a hyaline cytoplasm 
with an entire margin and with one or more inner bodies (nuclei) having a more 
or less complicated and regular structure. There do not appear to be any 
vacuoles in the smear preparations but in the tissue specimens vacuoles are 
quite distinct (probably artefacts). Most of the bodies show chromatoid 
granules of varying size and number and arranged about the nucleus like a ring. 
In stained specimens these granules are seen as basophilic granules, rods or 
circles. They are, therefore, properly classified as protozoan parasites. 

To demonstrate these Negri bodies in the brain cells of a suspected animal, 
the following method of Williams and Lowden 7 is recommended for diagnostic 
purposes. A fragment of the gray substance from the cerebral cortex in the 
region of the fissure of Rolando, another from Ammon's horn and a third from 

1 See Hamburger, Jour. Am. Med. Assn., 1912, LIX, 1590; Sutton, Ibid., 1913, LX, 115; 
Chipman, Jour. Cutan. Dis., 1912, XXX, 339; Adams, Journal Lancet, 1912, XXXII, 395; 
Jour. Am. Med. Assn., 1913, LX, 1784; Taylor, Ibid., 1913, LX, 1142; Davis, Jour. Infect. 
Dis., 1913, XII, 453; Hamburger, Illinois Med. Jour., 1914, XXV, 99; Dominguez. Med. 
Record, 1914, LXXXV, 608; Davis, Jour. Infect. Dis., 1914, XV, 483; Sutton, Jour. Am. 
Med. Assn., 1914, LXIII, 1153; Meyer and Aird, Jour. Infect. Dis., 1915, XVI, 399; Davis, 
Ibid., 1915, XVII, 174; Campana, Riforma Med., 1915, XXX, 925. 

2 Jour. Exper. Med., 1913, XVIII, 314. See, also, Levaditi~ C. R. Soc. de Biol., 1913, 
LXXV, 505; Kraus and Barbara, Deutsch. med. Wchnschr., 1914, XL, 1507; Noguchi, Berl. 
klin. Wchnschr., 1914, XLI, 193 1; Proescher, New York Med. Jour., 1914, C, 953. 

sZtschr. f. Hyg., 1903, XLIII, 507; XLD7, 520; 1909, LXIII, 421. 

4 C. R. Acad. d. so, 1879, LXXXLX, 444. 

5 Ibid., 1881, XCn, 159. 

8 See Babes, Traite de la rage, Paris, 1912: Proescher, Berl. klin. Wchnschr., 1913, L, 633; 
Poor and Steinhardt, Jour. Infect. Dis., 1913, XII, 202; Luzzani, Ann. de l'Inst. Pasteur, 
1913, XXVII, 907 and 1039; LeComte, Jour. Am. Med. Assn., 1914, LXIII, 1658. 

7 Jour. Infect. Dis., 1906, III, 452. 



PARASITES. 173 

the cerebellum, is taken from a section made at right angles to the surface and 
placed on slides about 1 inch from their ends. A cover-glass is now pressed 
upon it until it is spread out in a moderately thin layer; the cover-glass is moved 
slowly and evenly over the slide, leaving the first portion of the slide clean. 
Use slight pressure in making the smear, pressing rather more on the edge of 
the cover-glass away from the end of the slide toward which the cover-glass is 
moving, thus driving more of the nerve tissue along the smear and producing 
more well-spread nerve cells. Dry in air and stain as follows. 

Fix the air-dried smears in methyl alcohol for five minutes. Pour on the 
slide the staining solution and allow to stand for one-half to three hours. The 
staining solution is made by adding 10 drops of Giemsa's stain to 10 c.c. of dis- 
tilled water made alkaline by the previous addition of 1 drop of 1 per cent, 
potassium carbonate solution. The longer period of staining is preferable, as 
overstating does not occur even after 24 hours. Pour off the stain, wash in 
running tap- water for one to three minutes and dry with filter paper. If the 
smear be thick, dip it in 50 per cent, methyl alcohol before it is washed in water. 

The cytoplasm of the Negri bodies stains blue, while the central bodies 
and chromatoid granules stain a blue-red or azur. The larger bodies have 
usually a somewhat darker blue tint than the smaller ones. The cytoplasm of 
the nerve cells stains blue, while the nuclei are red, the nucleoli are a dull blue 
and the red cells a pink-yellow. 

If it is desired to make a quick examination proceed as follows, although 
this method is not to be advised. Fix in methyl alcohol for five minutes and 
add equal parts of Giemsa's stain and distilled water for ten minutes. Wash, 
dry and examine. 

Mallory 1 recommends the following technique. The smears made as above 
described are fixed in Zenker's solution for one-half hour; after being rinsed in 
tap-water they are placed successively in 95 per cent, alcohol, containing suffi- 
cient iodine to make a port-wine color, one-fourth hour, 95 per cent, alcohol 
one-half hour, absolute alcohol one-half hour, 10 per cent, aqueous solution of 
eosin for 20 minutes, rinse in tap-water, place in alkaline methylene-blue solu- 
tion for 15 minutes, differentiate in 95 per cent, alcohol from one to five 
minutes, dry with filter paper and examine. The cytoplasm of the Negri 
bodies is a magenta, the central bodies and chromatoid granules are a very 
dark blue, the nerve cell cytoplasm a light blue, the nucleus a darker blue and 
the red cells a brilliant pink. 

Van Gieson 2 recommends the following: To 10 c.c. of distilled water add 
3 drops of a saturated alcoholic solution of rose-aniline violet and 6 drops of 
LoefTler's methylene-blue solution. Fix the smears in methyl alcohol, pour 
the above stain on them and warm until steam rises. Pour off the stain, rinse 
in water, and allow to dry. The stain must be prepared fresh each time as it 
is not permanent. The cytoplasm of the bodies is a deep and distinctive red, 
their inner structures are a dark blue, the nerve cell light blue and the blood 
cells a pale salmon red. 3 

1 Pathological Technique, Philadelphia, 1911. 

2 Centralbl. f. Bakteriol., I. Abt., 1907, XLIII, 205. 

3 See Kotsevaloff, Russky Vrach., 1914, XIII, 616. 



174 DIAGNOSTIC METHODS. 

Noguchi 1 has succeeded in cultivating these bodies from infected animals 
and has, in turn, reproduced rabies by inoculating cultures containing the granu- 
lar, pleomorphic, or nucleated bodies. He has, then, recovered the granular 
and nucleated bodies from the brain of the inoculated animals. The specificity 
of these bodies would seem, therefore, to have been definitely established. See, 
however, Moon 2 and Williams. 3 



BIBLIOGRAPHY. 

Carpenter. The Microscope and its Revelations. Philadelphia, 190 1. 
Daniels. Laboratory Studies in Tropical Medicine. Philadelphia, 1903. 
Emery. Bacteriology and Hematology. Philadelphia, 1905. 
Hyde and Montgomery. Diseases of the Skin. Philadelphia, 1902. 
Jackson. Tropical Medicine. Philadelphia, 1907. 
Oertel. Medical Microscopy. Philadelphia, 1902. 
Pusey. Diseases of the Skin. New York, 1907. 

1 Jour. Exper. Med., 1913, XVIII, 314. 

2 Jour. Infect. Dis., 1913, XIII, 232. 

3 Jour. Am. Med. Assn., 1913, LXI, 1509. 



CHAPTER VI. 

THE URINE. 

I. General Considerations. 

The examination of the urine is one of the most important features of 
clinical diagnosis. So constant are the physical and chemical properties of the 
normal urine that any marked abnormality is easily detected. The relation 
between the kidneys and the blood is so close that the kidneys soon excrete 
any abnormal substances which have found their way into the blood-current. 
For this reason we find in the urine the abnormal products of perverted metabolism 
of the system or of special organs. It is true that the urinary findings may be, 
in any special case, secondary to those of the blood or of clinical examination, 
but in such conditions we may detect substances in the urine, which put us on 
our guard against making a specific diagnosis or point out the way to a correct 
differentiation. More or less marked changes in the character of the urine 
will occur whenever a pathologic condition exists anywhere in the system. 
These changes may not always be sufficient to be of direct diagnostic value, 
but in many cases may settle a differential diagnosis. 1 

In the urine we find excreted the products arising from the metabolism 
of the various proximate principles, both of the tissues and the food. Knowing 
the intake of such material, we are able from our examination of the urine to 
judge of the manner in which the system is handling the material brought to it. 
In recent years the study of the metabolism in various conditions has been so 
extended that determinations which a few years ago were unusual are now 
matters of almost daily routine. An examination of the urine will frequently 
reveal the presence of irregular digestive and absorptive powers of the intestines, 
through the appearance of certain abnormal products of protein decomposition. 
Moreover, the study of the nitrogen partition of the urine is taking on increasing 
importance from day to day, so that the estimation of the factors determining 
this division should be possible by any one attempting to follow the metabolic 
activity of the system in any specified condition. 

When the oxidative powers of the system are lessened, we find the urine 
showing abnormal products as an indication of such deficiency. These products 
are more or less characteristic and may be determined with a great degree of 
exactitude. Thanks to the work upon metabolism in diabetes, for instance, 
we now know that the glycosuria is clinically not of as great importance as is 
the presence of many other abnormal products associated with the sugar; in 
other words, a glycosuria must not be considered as identical with diabetes. 

1 See Freeman, Jour. Am. Med. Assn., 1914, LXIII, 1802; Diner, New York Med. Jour., 
1915, CI, 1007. 

175 



176 DIAGNOSTIC METHODS. 

The system has a definite disintoxicating power toward certain noxious 
substances, whether introduced from without or formed within. While the 
blood is of special importance in such processes as far as bacterial products are 
concerned, an examination of the urine will frequently reveal much information 
regarding the metabolic toxihs or the medicinal poisons. The estimation of 
the conjugated glycuronic and sulphuric acids of the urine will throw much 
light on the degree of this activity. In this connection it may be mentioned 
that indican, a product of bacterial decomposition of protein in the intestinal 
canal, may be taken as a direct indicator of the degree of such decomposition, but 
that we must not assume that all of the conjugated acids have such an origin. 

Besides these general indirect points of interest, an examination of the 
urine will often reveal a direct anatomical lesion of the kidneys. Time was 
when we regarded the mere presence of albumin in the urine as indicative of a 
kidney lesion, but we know that a thorough clinical examination is necessary 
before a diagnosis is possible. Albumin may or may not mean kidney trouble 
and may even be purely physiologic. Too much stress can hardly be laid upon 
the necessity of closely associating the urinary findings in any condition with the 
clinical symptoms of the case. The writer will have much to say later regarding 
the various abnormalities of the urine, but he wishes to impress at this point 
the fact that no finding, no matter how abnormal it may seem, should be con- 
sidered absolutely pathognomonic, without taking into consideration the 
clinical manifestations of the case. An albuminuria, glycosuria, cylindruria, 
or pyuria may mean one thing at one time and another at a second period, so 
that the worker is cautioned against jumping at conclusions. The urine will 
yield much information, providing the worker knows how to interpret his 
findings. A laboratory worker must be cautious in his attitude and report 
his findings without any attempt at interpretation, unless he is aware of the 
clinical history of the case in point. 

The writer must refer to works on physiology for the various theories 
which have arisen from time to time regarding the mechanism of secretion of the 
urine. Suffice it to say at this point that the urine is excreted through the 
activities of the kidneys, the water and salts being secreted by the glomeruli, 
while the majority of the excretory products are eliminated by the vital or select- 
ive activity of the epithelium of the renal tubules. It is evident, therefore, 
that the physical and chemical characteristics of the urine will depend both upon 
the blood-pressure 1 within the capillaries and the rate of flow through these 
vessels as well as upon the condition of the secreting epithelium. 

Collection and Preservation of the Urine. 

In all urinary examinations in which quantitative relations are to be studied 
it is necessary that a portion of the total 24-hour specimen be examined. This 
should be thoroughly mixed and carefully measured. The composition of 
the different voidings is so variable that no definite idea regarding the elimina- 
tion can be gained from a single specimen. 

1 See Lawrence (Am. Jour. Med. Sc, 1912, CXLIV, 330), who shows that the amount of 
blood in a unit of time is of much more importance than the pressure. See, also, Leschke, 
Ztschr. f. klin. Med., 1914, LXXXI, 14; Folin and Denis, Jour. Biol. Chem., 1915, XXII, 321. 



THE URINE. 177 

If a mere qualitative examination is to be made, a single specimen may be 
studied. In chronic nephritis, for instance, the morning urine may show points 
of interest as compared with that voided in the 'evening after a day's activity. 
In diabetes it may be desired to study the effect of a carbohydrate meal upon the 
sugar excretion. This may best be done by an examination of the urine passed 
three or four hours after such a meal. The microscopic examination is best 
made as soon as possible after voiding, but it is to be remembered that much 
variation may be noted in the sediment of the different voidings. 

In making the 24-hour collection, the patient is instructed to empty his 
bladder at a specified time, preferably at 7 a. m. This portion is thrown away 
and all urine passed from that time until the bladder is emptied at 7 a. m. the 
next day is saved. If one desires to separate the day and night urine, the 
voidings from 7 a. m. to 7 p. m. may be kept in one container and those from 
7 p. m. to 7 a. m. in a second vessel properly labeled. 

A thoroughly clean bottle of one-half to one gallon in capacity should be 
used as the container. 1 This should be well corked after each addition of urine 
and kept in a cool place. As urine undergoes decomposition more or less readily, 
depending upon bacterial activity, some preservative should be added to prevent 
such processes. The writer is accustomed to advise the use of a slight excess 
of chloroform. This may be removed by heating the urine and will not then 
interfere with the later reactions. If not removed, its presence will lead to a 
pseudocarbohydrate reaction. Three or four drops of formalin may be added 
for each pint of urine. This is an efficient agent, but it will lead to reactions 
simulating those for sugar and even albumin, and, moreover, will introduce a 
crystalline compound of formalin and urea into the sediment as well as 
markedly interfering with the bile, urobilin 2 and indican tests. Thymol may be 
added, but this may give a reaction similar to those for bile pigments, indican 
and albumin. Camphor, 3 chloral, and boracic acid have been used, but do not 
possess any virtues over the other preservatives mentioned. In any case, the 
worker should be on his guard in reporting abnormal findings without convincing 
himself that the reaction is not due to an added preservative. 

II. Physical Properties. 

(1). Quantity. 

The amount of urine passed within 24 hours depends upon several factors 
and varies both for individuals and for different races of people. It is self- 
evident that under normal conditions, the amount of urine will vary with the 
quality and quantity of the substances to be excreted, the condition of the renal 
parenchyma, the pressure and rate of flow of the blood-current, the vasomotor 
disturbances, the stage of digestion, the loss of fluid in the perspiration as 
influenced by the surrounding temperature, amount of exercise, and extent of 
fluid intake; upon the weight of the subject, the sex, and age. 

1 See Folin and Denis, Arch. Int. Med., 1915, XVI, 195. 

2 See Hausmann, Deutsch. med. Wchnschr., 1913, XXXIX, 1685; Schmiz, Ibid., 1914, 
XL, 128. 

3 See Rosenbloom, New York Med. Jour., 1914, XCIX, 735. 



178 DIAGNOSTIC METHODS. 

As a rule, the quantity of urine excreted varies between 1,200 and 1,500 c.c 
(40 to 50 ounces), reaching a maximum two or three hours after a large fluid 
intake. Women excrete somewhat less than men, while children void relatively 
more than do adults, although the actual amount is less. In the adult we find 
the amount of urine is almost directly proportional to his weight, a normal large 
individual excreting nearer 1,800 than 1,500 c.c, the amount being about 1 c.c. 
per kilo and hour, while with a child the excretion is about 4 c.c. per kilo and 
hour. 

The physiologic limits of the urinary excretion are about 750 and 3,000 c.c. 
In cases showing as high an output as 3,000 c.c. one is justified, perhaps, in 
assuming the presence of some pathologic condition. The kidneys are not 
easily deranged by the excess work put upon them in excreting large 
quantities of urine, so that we may find a secretion of many liters per day 
continuing for an extended period without endangering the normality of the 
excreting organ. 

Normally, the amount of urine excreted during the day by far exceeds that 
voided during the night, while the afternoon urine is usually more than that 
of the morning. We find, however, that in edematous conditions either of 
hepatic, cardiac, or renal origin, the night urine usually exceeds that of the day. 
This condition is known as nycturia. As it is, perhaps, more frequently asso- 
ciated with cardiac insufficiency, it may have a diagnostic importance in these 
cases. 

Polyuria. 

By this is meant an excretion of an increased amount of urine. Just 
what amount of urine is to be considered as indicative of polyuria will depend 
much upon the habits of the patient as regards daily intake of fluid. As a rule, 
anything above 2,500 c.c. is at least suggestive of this condition. 

Just what factors are to be held accountable for the polyuria, is not always 
easy to decide in every case. An increased intake of fluid together with an 
increased general blood-pressure will cause both an increased local renal 
pressure and an increased blood-flow through the kidney. While such a 
polyuria rarely exists in cases of ordinary chronic or active renal hyperemia, we 
find under the influence of drugs that a very decided increase in the urinary 
output may occur. 

A polyuria is observed in the convalescent stages of acute nephritis, in 
both chronic parenchymatous and interstitial nephritis, and in amyloid degenera- 
tion of the kidney. This excretion, especially in the chronic interstitial type, 
may be one in which the total solids are normal or reduced, and is then known as 
hydruria. 

Diabetes mellitus is more frequently, perhaps, than other conditions 
associated with a polyuria. The quantity eliminated is dependent both upon 
the increased intake as a result of the polydipsia as well as upon the dehydrating 
powers of the sugar. A certain relationship exists between the amount of fluid 
and the sugar, the polyuria being usually diminished by measures which decrease 
the amount of sugar excreted. This polyuria is not necessarily continuous and 



THE URINE. 179 

may alternate with periods showing a normal or subnormal amount of urine. 

In cases of diabetes insipidus we find the daily excretion of as much as 
50 liters or more of urine. According to Meyer, this polyuria is due to the 
attempt on the part of the kidneys to secrete sufficient water to hold the solids 
in solution. 1 

In cases associated with abnormal accumulations of fluid, such as pleuritis, 
ascites, and general edema, a polyuria will exist at the time of absorption of the ex- 
udates, owing to the presence of such large amounts of fluid in the blood-vessels. 2 

The so-called "epicritic polyuria " is frequently observed during convales- 
cence from acute febrile attacks. This is probably indicative of the elimination 
of toxic products which have accumulated in the system during the progress 
of the disease. It is supposed to be of favorable import when occurring in a 
febrile condition, but it is to be recalled that this polyuria may be followed 
by a later oliguria which is of grave significance. As a rule, however, it 
may be said that as the case improves th e urine is increased in amount. 

Polyuria may be observed in many nervous conditions, both functional 
and organic. 3 The cause is probably some disturbance of the vasomotor appa- 
ratus as a result, perhaps, of irritation of the floor of the fourth ventricle, 
cerebellum, or cord. Hysteria, neurasthenia, epilepsy, and chorea are fre- 
quently associated with a polyuria. A paroxysmal polyuria in the course of a 
suspected nervous disease is more indicative of a functional derangement, while 
a continuous polyuria is more frequently associated with true organic disease. 

Oliguria. 

This is a condition characterized by the excretion of a diminished amount 
of urine, 800 c.c. being given as the lower normal point of the urinary output. 
Here, again, the absolute figure must depend upon the patient and upon his 
customary excretion. A single examination is not sufficient to decide whether 
or not an oliguria exists. 

This condition is found, perhaps, most frequently in cases of broken 
compensation of the heart, where the blood-pressure is markedly diminished. 
It is present whether the cardiac incompetency be primary or secondary to 
hepatic, renal, or pulmonary lesions. 

Oliguria is noted in practically all acute febrile disorders, especially in 
typhoid fever. This is due, probably, to a combination of cardiac weakness with 
the increased loss of water by the skin and lungs. Moreover, we may have, in 
such states, a retention of fluid along with a direct contraction of the renal vessels. 

Acute nephritis as well as chronic parenchymatous nephritis are associated 
with a more or less extensive oliguria. This condition is probably referable 

1 See Lewis and Matthews (Trans. Chic. Path. Soc, 1913, IX, 16), who show the close rela- 
tion of hypersecretion of the pars intermedia of the hypophysis to diabetes insipidus. See 



Mi 



Socin, Ztschr., f. klin. Med., 1913, LXXVIII, 294; also, Forschbach, Ibid., 1913, LXXYII, 
Goldzieher, Verhandl. d. deutsch. path. Gesellsch., 1913, XVI, 272; Berblinger, Ibid., 281: 
Simmonds, Munch, med. Wchnschr., 1914, LXI, 180; Hohlweg, Ibid., 927; Fitz, Arch. Int. 
Med., 1914. XIV, 706; Mathews, Ibid., 1914, XV, 451; Krikortz, Hygiea, 1915, LXXVII, 49- 

2 See Baehr, Deutsch. Arch. f. klin. Med., 1013, CIX, 417; also, Leuret, Jour, de med. de 
Bordeaux, 1913, LXXXIV, 669. 

3 See Pirondini, Jour. d'Urol., 1914; V, 461; Mathieu, Bull. Acad, de Med., 1915, 
LXXIII, 208. 



l8o DIAGNOSTIC METHODS. 

to diminished functional activity of the glandular elements as well as to increased 
resistance within the tubules. A bilateral diffuse lesion is always necessary 
to cause much oliguria, as the sound kidney, if the trouble be unilateral, will 
take on vicarious activity. The more acute the condition, the greater the degree 
of oliguria. 

Oliguria may also occur following the administration of an anesthetic, 
in connection with eclampsia, hysteria, or epilepsy, after the loss of large 
quantities of fluid by hemorrhage, diarrhea, or vomiting, in cases of portal 
obstruction as seen in acute yellow atrophy or hepatic cirrhosis, or in cases in 
which pressure is exerted upon the vascular system, especially the vena cava, by 
tumors. 1 
Anuria. 

This oliguria may, in almost any case, proceed to complete anuria, which 
may or may not be of vital significance. Cases of anuria do occur without any 
preceding oliguria, as shown in acute nephritis and in some cases of hysteria. 
Anuria, per se, cannot be held responsible, however, for the uremic symptoms 
so frequently associated with it, as it may persist for many days, 19 in a case of 
Adams, without any uremic signs. 

Anuria may be due to obstructive, reflex, renal, and prerenal causes. We 
may have an occlusion of the urinary passages 2 on one side and a reflex closure 
on the other. Tumors, prostatic hypertrophy, and toxic and nervous bladder 
disturbances may lead to a great degree of oliguria amounting, almost, to anuria. 

The so-called prerenal causes of anuria include scarlet fever, which may 
lead to a severe nephritis, phosphorous poisoning, action of ether and chloroform, 
collapse, ureteral and urethral calculus, and cholera. 
(2). Appearance. 

Freshly voided urine should be clear and transparent. Only the faintest 
trace of any turbidity should be normally present, except soon after a meal 
rich in vegetable food, when a distinct turbidity may be noticed due to the 
precipitation of the phosphates in the alkaline urine. 

When allowed to stand for a short time, a light cloud is noted which 
gradually settles to the bottom of the container in the form of the so-called 
" nubecula." This contains a few small granular cells and a few epithelial 
cells and is composed largely of mucus. 

On standing for a somewhat longer period, as for instance over night, 
at the ordinary temperature, distinct crystals of uric acid may separate and 
appear in the sediment. If the temperature of the urine is allowed to fall to a 
considerable extent during this period, a somewhat more marked turbidity 
will be produced owing to the precipitation of the acid urates. This sediment 
is particularly noticeable if a highly acid urine becomes very cold. 

If kept for a longer period at room temperature, or a shorter period during 
the warmer months, a diffuse cloudiness will appear, due to the precipitation 
of the phosphates, owing to the lessened acidity or abnormal alkalinity of the 

1 See Cohn, Berl. klin. Wchnschr., 1915, LIT, 20S. 

2 See MacNider (Jour. Med. Research, 1912, XXVI, 79) for a discussion of the relation 
of swelling of the tubular epithelium to the urinary output. 



THE URINE. l8l 

urine. This alkalinity is due to the decomposition of the urea into ammonium 
carbonate. The crystals in this alkaline urine will be triple phosphates, 
calcium phosphates, ammonium urate, and calcium carbonate. 

Even before the urine becomes alkaline, a diffuse cloudiness may be 
present, due to the development of numerous saprophytic bacteria. This 
bacterial cloud is removed only with the greatest difficulty, as filtration of the 
urine has practically no effect upon it. Frequently, the addition of lead acetate 
to the urine will produce a voluminous precipitate, which may carry down the 
bacteria and permit of their filtration. This procedure is, however, not to be 
recommended, as other substances, if present in small amounts, may be carried 
down and thus escape detection. 

If the urine is cloudy when freshly voided, the turbidity may be the result 
of the precipitation of phosphates through the alkalinity or it may indicate the 
presence of an organized sediment, such as casts, epithelial cells, blood, and pus. 

The normal urine shows but very little viscosity, differing little from 
ordinary water in this respect. In certain conditions we find a marked degree 
of viscidity, which becomes especially apparent on attempting to filter the urine. 
In cases of chronic cystitis the excretion of a large amount of mucus may make 
the urine ropy and gelatinous. This increased viscosity may also be seen in 
cases of pyuria associated with decomposition. 1 

(3). Color. 

The color of the urine varies normally between various shades of yellow, 
the depth of color depending upon the concentration or specific gravity of 
the specimen. While the color is usually much paler in the urines of low 
specific gravity and very dark in those of high density, we find in diabetes 
mellitus a very pale urine with a high specific gravity. In cases of anemia 
the urine is always paler than normal, but in pernicious anemia the urine 
is highly colored owing to the marked destruction of the erythrocytes. 

As a rule, it may be said that an acid urine is more highly colored than 
an alkaline one, although many exceptions to this rule occur. There seems 
to be some difference between the urines passed at different periods of the 
day; thus the urine of the day is usually a distinct amber, while that of the night 
may take on a greenish tinge. To what this color in the latter case is due is 
at present unsettled. Several color scales have been introduced, such as those 
of Neubauer and Vogel and of Radde, but these are not sufficiently extensive 
to take in pathologic variations where they would be most important. As 
a rule, it is sufficient to divide the colors of the urine into those of the spectrum, 
making allowance for light, medium, and dark shades of each color. 

The pigments causing the normal and abnormal colorations of the urine 
will be discussed in detail in a later section. At this point the writer would 
say that normally these pigments are urochrome, uroerythrin, and urobilin, 
while the various conjugated glycuronic and sulphuric acids, blood pigments, 
biliary pigments, melanin, etc., are found in pathologic conditions. 

1 See Steensma (Nederl. Tijdschr. v. Geneesk., 1914, LVIII, 24), who reports fluorescence 
in certain types of urines. 



l82 DIAGNOSTIC METHODS. 

Pathologic Colorations. 

Deviating from the rule that the higher the specific gravity the more 
intense the color, diabetes mellitus shows an extremely light color with 
a high specific gravity. Owing to the lack of pigment, in chlorosis we find 
a very pale urine. Chronic interstitial nephritis and amyloid degeneration 
of the kidneys are associated with an extremely pale urine. 

In febrile conditions the coloration may range from an orange-red to a 
distinctly red tint, owing to the increase in the amount of urobilin. This 
deep color is especially noticeable in cases of severe pneumonia. 1 This reddish 
urine may also be due to an increase in the amount of uroervthrin, which 
is responsible for the deep color of the urate sediment so frequent in the concen- 
trated urines of febrile conditions, circulatory disturbance of the liver, and 
in cases associated with profuse perspiration. 

In cases of jaundice the urine may vary from a dark yellow or green 
tp a brown or black, depending upon the concentration of the urine, the amount 
of bile pigments, and upon certain chemical activity which occurs in such 
urine. Not only will the color of the urine be deeper, but the foam which 
appears on shaking the specimen will take on a distinct yellowish -brown tint. 
If this biliary urine be allowed to stand in the cold for some time, crystals 
of bilirubin may separate out and be seen in the sediment. 

Urine which contains blood may have a violet shimmer, may appear 
smoky, blood red, brownish-black, or even deep black in color. These varia- 
tions depend both upon the amount and kind of pigment present. Hemo- 
globin gives a more reddish tint to the urine, while methemoglobin produces a 
brownish shade. Such urine is always cloudy, owing to the admixture of 
corpuscles and other organic material. The blood found in the urine may 
arise from any point in the genitourinary tract or may be of systemic origin. 
In the latter case conditions which give rise to hemolysis will cause the appear- 
ance of hemoglobin in the urine. 

The condition of chyluria is characterized by the presence of large numbers 
of highly refractile globules of fat along with many morphological constituents. 
This gives rise to the appearance of a milky urine and is especially characteristic 
of infection with the filaria. It is not infrequent to find, in certain cases of 
hysteria, a specimen of milky urine, owing to the fact that the patient has 
added milk to the urine before sending it to be examined. The presence 
of a large quantity of pus will also give the urine a milky appearance. 

The urine of patients suffering with melanotic tumors may be perfectly 
clear when freshly voided, but becomes black or dark brown on exposure to 
the air. This reaction is due to the transformation of the pigment melanogen to 
melanin, and may be hastened by the addition of oxidizing agents to the urine. 
This darkening of the urine extends characteristically from above downward. 

The condition known as alkaptonuria, which is characterized by the ex- 
cretion of homogentisic and uroleucic acids, gives rise to the passage of a urine 
which is brownish-black in color and may be syrupy in consistency. This color 
is not always evident in the fresh specimen, but appears soon after being voided. 
1 See Hildebrandt, Ztschr. f. klin. Med., 19 11, LXXIII, 189. 



THE URINE. . 183 

In cases of peritonitis, suppuration anywhere in the system, gangrene, 
and marked intestinal putrefaction, the urine is frequently dark colored owing 
to the passage of certain aromatic products of decomposition, either indican 
or various derivatives of phenol. The coloration in these cases may vary from 
a dark brown or greenish-black to a distinct blue. These urines differ from 
those containing melanin in the fact that ferric chlorid does not blacken 
the urine as it does in the presence of melanin. This urine may contain a 
distinct amount of indigo, although the substance present is usually a different 
oxidation product. If indigo be present, a bluish-black scum will frequently 
rise to the surface of the specimen. 

Medicinal Coloration. 

After the use of carbolic acid either internally or externally, guaiacol, 
creosote, resorcin, naphthalin, salol, and various tar preparations the urine 
may vary from a dark brown to a black color. This coloration is due to the 
excretion of hydroquinon and of pyrocatechin, and may be evident only on 
allowing the urine to stand for some time. The urine containing pyrocatechin 
may reduce alkaline copper solutions, but will not affect such bismuth prepa- 
rations. While a dark brown or black coloration of the urine may be found 
in cases of hemorrhage, melanosis, malaria, alkaptonuria, ochronosis, and 
chronic tuberculosis, one should be on his guard, as the medication of the case 
may be responsible for such coloration. 

Methylene blue will color the urine a greenish to deep blue shade, which 
may last for several days. Usually within an hour after this drug is taken the 
urine may show a faint tinge of green which may be more clearly brought 
out by acidifying with acetic acid and warming. 

The use of the hypnotics, trional, sulphonal, and tetronal, frequently 
gives rise to the voiding of a urine which has a deep red-wine color, due to the 
presence of hematoporphyrin. Pyramidon produces a urine of rose-red color, 
the pigment of which is soluble in ether, chloroform, and amyl alcohol. Anti- 
pyrin and purgatin both produce distinctly red urines. Chrysarobin, 
senna, rhubarb, cascara, and santonin produce a golden-yellow urine which 
becomes red in the presence of alkali. This coloration is due to the excretion 
of chrysophanic acid. According to Gorup-Besanez, the pigment of beets, 
huckleberries, blackberries, etc., may under certain conditions be excreted 
in the urine and color it the corresponding shades. 
(4). Odor. 

The normal urine usually has a distinct aromatic odor which very much 

resembles that of beef broth. This odor is due to the presence of certain 

volatile acids and is more marked in urines of high concentration. 1 If the urine 

undergoes decomposition either within the bladder or on standing, a so-called 

"urinous odor" appears which is due to the decomposition of protein material. 

This odor is very markedly ammoniacal. Should such an odor appear in the 

freshly voided specimen, it is evidence of marked cystitis. 

1 Dehn and Hartman (Jour. Am. Chem. Soc, 1914, XXXVI, 2118 and 2136) believe the 
odor of urine to be due to a peculiar substance which they call "urinod." See, also, Hartman, 
Arch. Int. Med., 1915, XVI, 98. 



184 • DIAGNOSTIC METHODS. 

Abnormal decomposition of the urine, as evidenced by changes in the odor, 
may be found in conditions associated with decomposition of pus and may 
be due to the presence of hydrogen sulphid along with the ammonia. This 
condition may be observed in cases of perforation of an abscess into the urinary 
tract, in which case the urine may have a distinctly fecal odor if the intestine 
be involved, while in carcinoma of the bladder this repulsive odor of the urine 
may also be noticed. 

A distinct fruity odor is often present in cases of diabetes mellitus, in 
many febrile conditions, and in some stomach and intestinal troubles, which 
may be directly traceable to the presence of acetone. 

Certain medicaments, such as oil of turpentine, give rise to a distinct 
odor of violets in the urine. Menthol causes an odor of peppermint, while 
cubebs, copaiba, sandal-wood oil, tolu, and saffron produce a peculiar spicy 
odor. Valerian and asafetida are excreted as such in the urine and produce 
their characteristic odor. Certain foods, such as meat, bouillon, and coffee, 
produce a slight odor of the urine, while asparagus gives a peculiar char- 
acteristic odor due to the presence of methyl mercaptan. 
(5). Reaction. 

The normal urine has an acid reaction. According to the views formerly 
held, this acidity was directly due to the presence of acid salts, especially to 
sodium dihydrogen phosphate (NaH 2 P0 4 ), and not to the presence of any 
free acid. 

The recent work of Folin has shown that the phosphates in the clear urine 
are all of the monobasic (diacid) type. His figures indicate that the acidity 
of normal clear urines is ordinarily greater than the acidity of all the phosphates 
present and that the excess must be due to free organic acids. For this reason 
the methods of Freund and of Lieblein for the determination of the acidity 
of the urine must be given up. To quote from Folin : 1 "The current attractive, 
and in a measure plausible, belief that the acidity of urine is regulated by 
variations in the relative proportion of the two forms of 'acid phosphates' is, 
therefore, erroneous. If urine does at no time contain comparatively strong 
acids in the free form, the reason is in part the variability of the ammonia 
formation and in part the presence of salts of organic acids. In a mixture of 
salts containing an excess of acids it is the weakest which will remain uncombined 
and the strongest organic acids will, therefore, exist as salts; but if the total 
amount of acidity becomes abnormally great, the quality (the strength) of the 
free acids may change." 

From the standpoint of physical chemistry the acidity of the urine, as of 
all other acid solutions, should represent the absolute number of dissociated 
hydrogen ions in a definite quantity of the urine. We are, therefore, face to 
face with the same problem confronting us in the examination of the alkalinity 
of the blood. In the case of the urine the question of indicator to be used in 
the titration test is a matter of great moment, as no two indicators will give 
the same degree of acidity. The one naturally to be selected would be that 
which will react to every possible substance of an acid nature. 
1 Am. Jour. Physiol., 1905, XIII, 45. 



THE URINE. 185 

If we use the methods of physical chemistry we find, according to Hober, 1 
that the urine is only about 30 times as acid as is distilled water and only about 
one ten-thousandth as acid as the titration figures would indicate. Such being 
the case, we must either entirely revise our figures for the acidity of the urine 
or employ methods which can be more easily carried out by the general worker 
than can those of physical chemistry. 2 

Folin has, therefore, introduced a method which uses direct titration 
of the urine and employs phenolphthalein as an indicator. This indicator 
reacts to all bodies of an acid nature, but cannot overcome certain difficulties 
which are in the way of direct titration. These obstacles are (1) the occur- 
rence of calcium in the urine in the presence of the monobasic phosphates, and 
(2) the presence of ammonium salts. He has found that the addition of potas- 
sium oxalate to the urine will do away with these difficulties by holding in 
solution both the di- and tri-calcium phosphates and by preventing the dissocia- 
tion of the ammonium compounds. 

Folin's Method. 
Total Acidity. 

Twenty-five c.c. of urine are treated with 15 to 20 grams of powdered 
potassium oxalate and one or two drops of a 1 per cent, alcoholic solution of 
phenol-phthalein. The mixture is shaken rapidly for one or two minutes 
and titrated at once with a tenth-normal sodium hydrate solution until a 
faint, distinct, permanent pink color is obtained. It is advisable to shake 
the flask during the titration so as to prolong the effects of the potassium 
oxalate. The acidity is expressed in terms of the amount of tenth-normal 
sodium hydrate solution necessary for neutralization of the 24-hour amount of 
urine. This is expressed as T, which is, on an average, 617. 

Free Mineral and Organic Acidity. 

Determine the amount of total phosphates present by titration with 
uranium nitrate solution as described later. Seven and one one-hundredth 
mg. of P 2 O s have an acidimetric value equal to 1 c.c. of tenth-normal acid. 
The total acidimetric value of the phosphates of the 24-hour urine may be 
easily determined with the help of this factor, by converting the amount of 
phosphates into terms of N/10 acid. 

From the total acidity (T) subtract the acidimetric value of the phosphates 
(P). The remainder is the acidity due to uncombined organic acids, and 
the difference, that obtained from calculating all the phosphoric acid as di- 
acid phosphate, is the free mineral acidity. For all ordinary studies of the 
acidity of the urine the direct titration of the total acidity and of the phosphates 
gives the necessary information. 3 The excess of the total acidity above that 
calculated from the phosphates gives the total free acids present. If the 

1 Beitr. zur chem. Physiol, u. Path., 1903, III, 525. 

3 See Henderson and Palmer, Jour. Biol. Chem., 1913, XIII, 393; Ibid., 1913, XIV, 81; 
Arch. Int. Med., 1913, XII, 153; also, Henderson, Science, 1913, XXXVII, 389; Henderson, 
Palmer and Newburgh, Jour. Exper. Pharmacol., 1914, V, 449; Henderson and Palmer, Jour. 
Biol. Chem., 1914, XVII, 305; Ibid., 1915, XXL, 37; Ibid., 57; Arch. Int. Med., i9i5,XVI, 109; 
Host, Ztschr., f. klin. Med., 1915, LXXXI, 266. 

» See Walpole (Biochem. Jour., 1914, VIII, 628) for a discussion of various new indicators. 



l86 DIAGNOSTIC METHODS. 

acidity calculated from the total phosphates is greater than the titrated acidity, 
then there are practically no free organic acids present, and the titrated acidity 
represents the amount of phosphates present in the diacid form (Folin). 

While the acidity of the urine is best determined and expressed as outlined 
above, it seems wise to the writer to retain the same style of expression for the 
acidity as used in stomach analysis. With this nomenclature one would state 
the acidity of the urine in degrees; that is, the amount of tenth-normal sodium 
hydrate necessary to neutralize ioo c.c. Under normal conditions this will 
vary from 35 to 45 . It may be increased by a diet rich in meat, while it is 
decreased by a vegetable diet. There are many acids produced in the oxida- 
tion of protein, among which we find sulphuric, phosphoric, uric, and the 
oxyaromatic acids. Ordinarily, these play an indirect part in the acidity of 
the urine, although this phosphoric acid may exist in part as the dihydrogen 
phosphate and in consequence increase the acidity of the urine. The regulation 
of the metabolism is such that an increase of the acids produced in the system 
or taken into it from without is neutralized by an increased formation of am- 
monia, the salts appearing in the urine as the ammonium salts which do not, 
of course, increase the acidity of this fluid. This is the basis upon which one 
estimates the amount of ammonia in following a condition of acidosis. 1 

The reaction of the urine varies at different times of the day. The acidity 
appears to be highest in the morning before breakfast and is diminished after 
a meal, due to the secretion of hydrochloric acid into the stomach. The reaction 
of the urine may even be alkaline for a period of two or four hours after each 
meal, in which case the urine will be turbid from the precipitation of phosphates. 
This reaction of the urine following meals is known as the ''alkaline tide" 
of the urine. Between meals the acidity of the urine will gradually increase 
until the next meal is taken. 

The reaction of the urine is modified to a great extent by the use of drugs. 2 
Thus, alkalies, such as carbonate and bicarbonate of sodium, will render 
the urine alkaline if taken between meals, while if taken just preceding a meal 
they will be neutralized by the gastric juice. All organic acids of the fatty 
series are oxidized in the system to carbonic acid and combine with bases 
forming basic salts which render the urine alkaline or less acid, providing 
these acids are not taken above the point of tolerance, as the writer has shown 
that large doses of such acids as citric acid will increase the acidity of the urine. 
The mineral and aromatic organic acids will, however, practically always 
increase the acidity. 

In many pathologic conditions we find the reaction of the urine variable. 3 
Abnormal gastric activity may be either associated with an increase or a 

1 Fischer (Nephritis: An Experimental and Critical Study of Its Nature, Cause and the 
Principles of Its Relief, New York, 1912) believes that the abnormal production or accumulation 
of acid in the cells of the kidney and the action of this acid on the colloidal structures of the 
kidney are accountable for all the changes which characterize nephritis. 

2 See de Jager (Biochem. Ztschr., 1912, XXXVIII, 294), who shows that magnesium sul- 
phate increases the acidity, while sodium sulphate diminishes it. 

3 See Newburgh, Palmer and Henderson, Arch. Int. Med., 1913, XII, 146; also, Pertik 
(Virchow's Arch, f . path. Anat., 19 13, CCXIII,465) who&nds a decreased acidity in tuberculosis; 
Labbe and Vitry, Presse Med., 1914, XXII, 437. 



THE URINE. 187 

decrease in the acidity of the urine, depending upon a condition of hypo- or 
hyperacidity of the gastric juice. The rapid absorption of a transudate or 
exudate will lead to the excretion of an alkaline urine from the presence of an 
increased amount of alkaline salts. An alkaline urine is not infrequently 
seen after intestinal hemorrhage, in certain cases of pneumonia, typhoid fever, 
chronic nephritis, and in cases in which exudates from the urinary tracts have 
become mixed with the urine. In certain cases of nervous diseases and in 
some cases of anemia we may also find an alkaline urine. The urine in all 
of the above cases will show, if tested by litmus-paper, an alkaline reaction in 
which the bluing of the red litmus-paper is permanent. This condition is 
known as fixed alkalinity and is quite distinct from the following type. 

In cases of decomposition of the urine within the urinary tract, through the 
influence of bacteria, the urea is decomposed into ammonium carbamate 
and carbonate. The alkaline reaction of the urine in such cases will be 
shown by a blue color of the red litmus-paper either held above it or placed 
in it, the blue color disappearing when the paper is dried. This condition is 
known as volatile alkalinity. If the urine shows this volatile alkalinity on 
being voided, the finding is significant of trouble somewhere along the urinary 
tract, especially within the bladder. 

Benedict has recently introduced the term "acid unit" into the study of 
the urinary output. One c.c. of urine with an acidity of 1 degree is equivalent 
to 100 acid units. In other words, one may determine the acid units in the 
24-hour specimen by multiplying the number of cubic centimeters of the 
urine by the degrees of acidity. This is normally about 40,000. 

(6). Specific Gravity. 

The specific gravity of the normal urine ranges between 1,015 an d 1,025, 
with an average of 1020. This specific gravity will depend, of course, upon 
the amount of fluid intake, the quantity of the 24-hour specimen of urine, the 
degree of tissue activity, and the condition of the secreting organs. The 
intake of a large volume of water may reduce the specific gravity of the urine 
to a very low figure and, correspondingly, a small intake may lead to a urine 
of high gravity. We find, therefore, that perfectly normal urines may show 
specific gravities ranging from 1010 to 1030, with pathologic variations from 
1,002 as high as 1,060 or more. 1 

In general routine work it is essential that the specific gravity of the 
24-hour specimen be determined. Except in unusual cases, absolutely noth- 
ing of diagnostic value may be learned by the determination of the specific 
gravity of a single voiding of urine. The variations at different times of the 
day, under the influence of food, digestion, activity of the skin and lungs, 
and exercise, may be so great that apparent pathologic figures may be obtained 
from a single specimen. Such variations are overcome for the most part 
in the 24-hour specimen unless pathologic conditions are present to keep 
up such variations. In some cases, especially in chronic diffuse nephritis, 
the morning specimen of urine almost invariably has a lower specific gravity 

1 SeeFrey, Deutsch. Arch. klin. Med., 1912, CVI, 347. 



DIAGNOSTIC METHODS. 



than that of the other periods of the day. For this reason one may determine 
the specific gravity of a single specimen of such urines. 

It is of especial importance that the total amount of urine in the 24-hour 
specimen be taken into consideration in judging of the value of a specific 
gravity. Thus in chronic interstitial nephritis we may find a large volume 
of urine with a low specific gravity, while in diabetes mellitus an even higher 
volume of urine may be present, showing a very high spe- 
cific gravity. 

Technic. 

The most accurate method of determining the specific 
gravity is, of course, the use of the pycnometer. The prin- 
ciple of this method is the determination of the weight of 
a definite volume of urine as compared with that of the 
same volume of distilled water under the same conditions 
of temperature and atmospheric pressure. This method 
will be discussed in the section on Blood, to which the 
reader is referred. 

The clinical method of estimating this factor is a dis- 
tinctly areometric one. The principle of this method is 
that a body immersed in a fluid will displace an amount 
of fluid equivalent to the loss in its own weight. By the 
use of instruments known as hydrometers or in the case of 
the urine as urinometers, this displacement is measured 
by immersing the hydrometer in the fluid and observing 
the point to which this instrument sinks. The stem of the 
hydrometer is graduated in divisions from 1,000 to 1,060 
by differences of i°, the 1,000 point being that to which the 
instrument sinks when immersed in distilled water at the 
temperature to which the instrument is calibrated. Any 
variation in the density of the solution in which this hy- 
drometer is immersed will be evident by the depth to which 
it sinks, the more concentrated the solution the less will 
the instrument sink. 
The vessel in which the urine is poured should be cylindrical in shape, 
with parallel sides and wide base and sufficiently tall to permit of the complete 
sinking of the hydrometer. The forms of this cylinder with fluted sides are 
perhaps more desirable than the plain cylinder, as the bulb does not tend 
to stick to the sides of the vessel so readily. 

The vessel is filled about four-fifths full of urine, any foam being re- 
moved by the use of filter-paper. The hydrometer is placed in the urine with 
a twisting motion and allowed to come to rest. The depth to which the 
stem is immersed is then read off by observing the mark which coincides 
with the lower meniscus of the urine as seen from below. The worker should 
never attempt to read the specific gravity from above as a slight meniscus 
interferes with the accuracy of his reading. The worker must be absolutely 




Fig. 70. — Urin- 
ometer and cylin- 
der. (Hawk.) 



THE URINE. 189 

sure that the urinometer neither rests upon the bottom of the cylinder nor 
touches the sides, but should see that it floats perfectly free in the urine. 

The temperature at which the reading is taken is a matter of some 
moment as some of these instruments are graduated at 15 C. The ordinary 
model, as made by Squibb, is graduated at 25 C, which is, perhaps, more 
nearly the working temperature of the room. A variation of 3 in tempera- 
ture between that of the room and that at which the instrument is calibrated, 
will give a difference of i° of specific gravity; that is, a difference in the fourth 
place of the specific gravity. In ordinary clinical work corrections for varia- 
tions in temperature are usually unnecessary, as variations of two or three 
points in the fourth place of the specific gravity are of absolutely no impor- 
tance, as such changes might be attributed to chemical variations on stand- 
ing, even though the most accurate methods of estimating the specific gravity 
were used. 

If the quantity of urine be very small, it may be diluted with distilled 
water, so that the measuring cylinder may contain enough material to permit 
of a density estimation. The specific gravity of this diluted urine is then 
determined as above and the last two figures of the specific gravity are multi- 
plied by the degree of dilution. 

Rough Estimate of Total Solids. 

As the degree of specific gravity is directly proportional to the amount 
of solids contained in the urine, one may roughly judge of the total solids 
by a simple calculation as follows: If the last two figures of the specific 
gravity be multiplied by 2.33 "(Haeser's coefficient), the result will be the 
approximate number of grams of total solids in every 1,000 c.c. of urine. Know- 
ing the quantity of urine passed in the 24 hours, a simple calculation will 
yield the total 24-hour excretion of solids. Long uses the coefficient 2.6. 
Instead of the above figure one may multiply the last two figures of the specific 
gravity by 1.1 (Haines' coefficient) and obtain the number of grains of solids 
in each fluidounce of the urine. This figure when multiplied by the total 
number of ounces of the 24-hour specimen will yield the excretion of solids 
in grains. This latter method has some advantage for the older practitioner 
who has not accustomed himself to the use of the metric system. 

It is to be said that neither one of the above methods can give anything 
but approximate results and in pathologic urines are absolutely unreliable. 
Highly albuminous urines invariably show a reduced specific gravity, while 
a high sugar content is associated with an increase in the density. In either 
one of these cases a calculation of the total solids by the above method will 
yield inexact figures. If the worker desires to know the exact amount of 
total solids in the 24-hour specimen, and this is sometimes advisable, recourse 
should be had to more exact methods of determination which will be discussed 
later. 

The specific gravity of a specimen of urine varies, of course, with the 
amount of total solids. 1 Normally, these range from 60 to 70 grams with a 

1 See Jacob, Deutsch. Arch. f. klin. Med., 1913, CX, 1. 



I90 DIAGNOSTIC METHODS. 

24-hour excretion of 1,500 c.c. of urine. The urea usually constitutes about 
one-half of the total solids. As the normal percentage of urea is approxi- 
mately two with a specific gravity of 1,020, the writer has been struck with 
the usual close relationship of the percentage of the urea to the specific gravity. 
In watching this point in over 2,000 examinations of urine within the last 
year, the writer has observed that the percentage of urea will practically parallel 
the last two figures of the specific gravity; in other words a specific gravity of 
1,015, for instance, will normally be associated with a urea content of 1.5 
per cent. This statement is true only in those cases which contain neither 
albumin nor sugar. 

As a rule, it may be said that the specific gravity of the urine is inversely 
proportional to the amount of fluid eliminated. It will, therefore, be evident 
that conditions leading to an oliguria will produce a high specific gravity, 
while those causing polyuria will give a low specific gravity. This state- 
ment must be modified when considering certain pathologic conditions, as 
we may find a diminished amount of urine with low specific gravity as in 
chronic nephritis in which the salts are diminished, although the organic albu- 
minous bodies are much increased; while in diabetes mellitus we have an abun- 
dant urine of high specific gravity. 

The specific gravity of the urine is of more or less importance in judging 
of the activity of the kidneys. In acute nephritis we find a urine of high 
specific gravity, while in the chronic types of renal disease the specific gravity 
is low owing to the diminution of the salts. It is to be noted in this connection 
that in the case of so-called "functional" albuminuria, the specific gravity 
of the urine is above the normal figures. A marked reduction in the specific 
gravity of any case of nephritis is of dangerous import. 

(7). Optical Activity. 

According to Haas, 1 the normal urine is slightly levorotatory, ranging 
from 0.01 to 0.18. This optical activity is due to traces of the conjugated 
glycuronic acids which will be discussed later. 

An increase in this levorotatory power is observed due to the presence 
of increased amounts of glycuronic acid, /3-oxybutyric acid, albumin (in 
amounts over one-half part per thousand), and levulose. Dextrorotatory 
urines depend upon the presence of glucose, maltose, and lactose, while the 
presence of pentose usually gives rise to an optically inactive urine or one 
at least showing only a slight degree of dextrorotation. 

This optical activity under the influence of pathologic products will be 
discussed in the section on Carbohydrates, to which the reader is referred. 

III. Chemical Properties. 

(A). Normal Composition. 

The tables given in most text-books showing the chemical composition 
of the urine cannot be regarded as absolutely indicative of the excretion as 
shown in the every-day specimens of urine. The composition of the urine 

1 Centralbl. f. d. Med. Wissensch., 1876. 



THE URINE. 191 

is absolutely dependent upon the diet under normal conditions, so that a 
table to be exact must embrace the findings under a specified diet. * Perhaps 
the most frequently quoted table is that of Parkes, which may be found in 
almost any text-book dealing with the urine. This table gives the various 
figures for the different substances excreted, but does not take into considera- 
tion the amount of the various types of foods used in the diet leading to this 
excretion. The writer, therefore, feels that it is wise to omit such a table at 
present, as we have no method of comparison of the excretion with the intake. 
The recent work of Folin upon the urine of persons both under a mixed diet 
and a nitrogen-free diet gives an " approximately complete" determination of 
the urinary constituents under absolutely fixed dietary conditions. The figures 
for such diets will be given under the head of each individual substance discussed. 

The daily urine of a healthy adult will vary between 1,200 and 1,500 c.c. 
in amount and will contain from 60 to 70 grams of total solids, of which the 
inorganic constituents form from 25 to 30 grams and the organic between 35 
and 40 grams. 2 The inorganic constituents consist of the phosphates of 
sodium, potassium, calcium, and magnesium, the chlorids and sulphates 
of the alkali metals, various types of ammonium salts, traces of nitrates, cal- 
cium carbonate (especially under vegetable diet), and traces of iron compounds. 
While these inorganic substances have not hitherto been credited with much 
importance, to-day we are realizing more and more that much is to be learned by 
a careful study of the inorganic excretion. 3 

The organic substances are of especial importance both in metabolic 
work and in the diagnosis of pathologic conditions. While we do not by any 
means know everything concerning the variations in excretion of these organic 
products, yet we do know much which is helpful in our direct diagnostic work 
as well as in our study of the progress of the disease. Among the organic 
substances which are more or less normal (although not always in large amounts) 
in the urine we find the lower and higher fatty acids, oxalic acid, acetone, 
glycerophosphoric acid, a trace of glucose, lactose (especially in nursing 
mothers), carbamic acid, urea, oxaluric acid, allantoin (especially a few days 
after birth), creatinin, uric acid, purin bases, thiosulphuric acid, tauro- 
carbamic acid, cystin, chondroitin-sulphuric acid, inosite, hippuric acid, 
benzoic acid, phenaceturic acid, p-oxyphenyl-acetic acid, hydro-p-cumaric 
acid, skatol-carbonic acid, conjugated sulphuric and glycuronic acids, oxy- 
proteic acid, pigments, organic iron compounds, traces of protein, and ferments. 

Under pathologic conditions we may find lactic acid, large amounts of 
acetone, aceto-acetic acid, /3-oxybutyric acid, fats, large amounts of glucose, 
levorotatory carbohydrates, r-arabinose, lecithin, cystin, putrescin, cadav- 
erin, ptomaines, oxymandelic acid, leucin, tyrosin, homogentisic acid, uro- 
leucic acid, cholesterin, cholic acid, glycocholic acid, tauro-cholic acid, various 

1 See Long, Jour. Am. Med. Assn., 1912, LVIIT, 757; also, Long and Gephart, Jour. Am. 
Chem. Soc, 1912, XXXIV, 1229. Kinloch, Jour. Path, and Bacterid., 1914, XIX, 77; Blather- 
wick, Arch. Int. Med., 1914, XIV, 409. 

2 See Atkins and Wallace (Biochem. Jour., 1913, VII, 219) for a discussion of the critical 
solution point of urine. 

3 See Baumann and Howard (Arch. Int. Med., 1912, IX, 665) for a discussion of the inor- 
ganic metabolism in scurvy. 



192 DIAGNOSTIC METHODS. 

derivates of phenol, hematin, hematoporphyrin, methemoglobin, other blood 
pigments, bile pigments, melanin, and protein material. 

(a). Total Solids and Total Ash. 

The estimation of the total solids of the urine is a matter of considerable 
difficulty, owing to the fact that evaporation of the urine leaves a syrupy 
residue which is dried only with much trouble and constant loss of ammonia, 
formed through the action of diacid sodium phosphate upon urea in concen- 
trated solution. One may come very close to accurate results by placing a 
weighed amount of dry clean sand in a weighed platinum dish and adding 
10 to 15 c.c. of urine. This is then evaporated upon the water-bath and 
later in the drying oven at 105 C. The dish is then placed in the desiccator 
and allowed to remain until the weight becomes constant. Knowing the 
weight of the dish and sand, the amount of the urine added, and the weight 
of the dish after the urine is evaporated the total solids may be easily calculated. 
In case a large amount of residue is desired for quantitative work, recourse 
may be had to the method of Slagle, 1 of adding 5 c.c. of concentrated H 2 S0 4 
to each liter of urine showing a specific gravity up to 1,020 and evaporating 
this to dryness. More H 2 S0 4 must be used with the more concentrated urines. 

The estimation of the ash is as follows: Fifty c.c. of urine are evaporated 
to dryness over the water-bath in a weighed platinum or porcelain dish. 
The dish is then heated, while covered, over the free flame until gases cease 
to be evolved, especial care being taken not to permit sputtering of the con- 
tents. In some cases it is possible completely to incinerate the urine by long- 
continued heat. However, a more usual procedure is to treat the carbonized 
residue with distilled water, thoroughly stir the mixture and filter through 
a filter-paper whose ash is known. The contents of the dish should be washed 
onto the filter several times and the material upon the filter also washed with 
boiling water. The filter-paper and its contents are now placed in' the dish 
and completely incinerated. After this procedure the filtrate and washings 
of the original carbonized material, which contain most of the inorganic con- 
stituents, are placed in the dish and evaporated at ioo° C. to dryness, and 
then incinerated over the free flame. The dish is now placed in a desiccator 
and dried to constant weight. Knowing the weight of the dish with its con- 
tents and the original weight of the dish a simple calculation will give the 
amount of ash in the 50 c.c. of urine taken. As originally stated the inorganic 
constituents will range between 25 and 30 grams under normal conditions. 

(b). Inorganic Constituents. 
(1). Chlorids. 

The chlorids are one of the most important groups of inorganic solids 
in the urine. They are derived entirely from the food and, in consequence, 
the amount of excretion will depend upon the intake. The chlorids actually 
forming constituent parts of the food exist in combination with potassium and 
calcium, while those which are added as seasoning to the food are practically 
always in the form of sodium chlorid. As the amount contained in the food 
1 Jour. Biol. Chem., 1910, VIII, 77. See also, Braman, Ibid., 1914, XIX, 105. 



THE URINE. I93 

is trivial in comparison to that added, we are accustomed to regard practically 
all of the intake as sodium chlorid. This is a constituent of the serum of the 
blood and of other tissues, while the potassium salt is in more direct relation 
with the cellular elements. 

Under normal conditions from 10 to 15 grams of sodium chlorid are 
eliminated in 24 hours. The administration of a diet rich in salts will in- 
crease this amount, while a salt-poor diet will diminish the amount up to a 
certain point. If the diet be a starvation one, or an absolutely salt-free one, 
the chlorids will disappear almost entirely from the urine. The regulation 
of the metabolic activity of the system is such that a certain amount of salt 
must be retained in order to preserve the osmotic equilibrium. For this 
reason we find that withdrawal of salt from the diet does not lead to any 
appreciable diminution of the normal chlorid content either of the blood or 
tissues. An increase in the elimination of salt is practically always followed 
by a retention of salt unless a sufficient supply is furnished by the food. If 
food containing sodium chlorid be given after a period of salt-free diet, a por- 
tion of this salt will be retained. Conversely, we find, if the body has for patho- 
logic reasons retained sodium chlorid, that an increased elimination will 
follow. This metabolic activity is intimately associated with the general 
protein metabolism of the body. Any increase in the amount of circulating 
protein as compared with the living protoplasm will be followed by an increased 
elimination of the chlorids, which have been previously retained by the living 
or active protein material of the protoplasm. This fact is shown by the rela- 
tion between the elimination of the chlorids and the total nitrogen. With 
an ordinary diet this ratio is as one to one, but in disease it may be much 
disturbed owing to chlorid retention through renal insufficiency. The blood 
and tissues of patients with nephritis show a higher chlorid content than 
those of normal individuals. We should, therefore, expect, if the law of 
increased chlorid excretion being dependent upon increased circulating 
albumin were to hold, that the chlorids would be increased along with the 
albumin in nephritis. But we find that the kidney under these circumstances 
is unable to excrete the increased amount of salts circulating in the blood. 

A further method of withdrawing sodium chlorid from the body con- 
sists in the administration of large quantities of alkaline carbonates or of 
compounds of the alkalies with vegetable acids. As soon as these are given 
the body becomes poor, not only in acid substances and in HC1, but also 
at the same time in sodium and potassium. Practically speaking, the body 
becomes directly impoverished in NaCl. The body may also lose chlorin 
when vomiting is frequent, when absorption is diminished, when the stomach 
is regularly washed out, and when diarrhea is marked. This loss may be 
especially noted in cases of hyperacidity of the gastric juice associated with 
vomiting. The chlorin in these conditions is withdrawn in the form of free 
acid, and in consequence the alkalinity of the tissues may be increased. 
Physiologic Variations. 

The amount of sodium chlorid excreted will depend directly upon the 
amount ingested. We may find as high as 30 grams of salt in the 24-hour 

13 



194 DIAGNOSTIC METHODS. 

specimen or it may be as low as 2 grams, salt-free diet reducing the elimination 
to a mere trace. The elimination may be increased by active exercise, by 
increasing the water intake and hence the water output, and by the intake 
of a large amount of vegetable food. Much more chlorid is apparently 
excreted during the day than during the night. 1 

Pathologic Variations. 

A marked diminution of the chlorids, which may in some cases be al- 
most complete, has been supposed to be pathognomonic of pneumonia. 
This, however, has been shown to be fallacious, as the same condition occurs 
in most acute febrile states, with a possible exception of intermittent fever. 
In a doubtful fever a large diminution in the amount of urinary chlorids 
might be strongly presumptive of pneumonia, but would be conclusive only 
in the presence of distinct clinical signs of this disease. While the retention 
of these chlorids in the exudate of pneumonia may partially explain the 
diminution in the urine, it cannot explain the fact that the chlorids of the 
food are also retained. The explanation is more likely to be found, in 
the writer's opinion, in an existing renal insufficiency. 2 This same retention 
of chlorids will be found also in any condition in which there is a transudate 
or exudate of any considerable bulk, so that this factor must play a certain 
role. As crisis approaches in pneumonia, the chlorids of the urine will in- 
crease in favorable cases, while in those of bad prognosis no such increase will, 
as a rule, be observed. Van der Bergh believes the explanation of the dimin- 
ished urinary chlorids in pneumonia to be an attempt on the part of the blood 
to maintain its osmotic pressure, the chlorids remaining fixed in the tissues 
owing to the increase of the products of metabolism in the plasma. 

The chlorids are diminished in all acute and chronic renal diseases 
associated with albuminuria. The work of Widal upon the influence of 
chlorids upon the progress of a renal disease has brought out the facts that 
not only do we have such a chlorid retention, but that the presence of chlorids 
in the food will increase both the albuminuria and the edema of these con- 
ditions. While these facts are incontrovertible, we must take into considera- 
tion, as Richter has shown, the amount of water intake as well. 3 This is 
such an important field to the clinician that the writer would refer to other 
works giving the details of the " dechlori&ization" treatment. 

A severe diarrhea will also diminish the amount of chlorids in the urine, 
as the chlorids of the food are carried off by the bowel too quickly to permit 
of absorption. In cases of carcinoma of the stomach, in dilatation either 
from hypersecretion or stricture of the pylorus, and in some cases of ulcer 
of the stomach a diminution or even total absence of chlorids in the urine may 
be observed. 

In most chronic diseases, in anemic conditions, in rickets, and in marked 
nervous diseases, such as melancholia or mania, the amount of chlorids may 

1 See Borelli and Girardi, Deutsch. Arch. f. klin. Med., 1914, CXVI, 216; Goldberg and 
Hertz, Ibid., 201. 

2 See Snapper, Deutsch. Arch. f. klin. Med., 1913, CXI, 281; also, Roff, Cor. — Bl. f . 
schweiz. Aerzte, 19 13, XLIII, 14 10. 

3 See Fischer, Jour. Am. Med. Assn., 1915, LXIV, 325. 



THE URINE. I95 

be greatly reduced. If the output of chlorids be very low in a chronic disease, 
the prognosis becomes grave unless the diet can explain the diminution. 1 

A diminution is observed in most febrile diseases, especially in the exanthe- 
mata, while in typhoid fever the reduction is not so marked. This slight diminu- 
tion in typhoid fever may serve as a distinguishing point in the diagnosis of men- 
ingitis from typhoid fever, in the former case the diminution being much more 
marked than in the latter. In acute yellow atrophy of the liver the chlorids 
are diminished, while in cirrhosis of the liver they are somewhat increased. 2 

The Chlorids are increased in all conditions which have previously shown 
a retention, according to the law which has been previously discussed. We 
find thus an increase in the period of convalescence from acute febrile diseases, 
especially pneumonia. Strangely enough, the chlorids are found markedly 
increased in diabetes insipidus, 3 which is associated also with the excretion 
of a large amount of urine. In epilepsy an increase may be observed follow- 
ing the attack. 

The chlorids are increased in the urine after the use of chloroform, 
whether administered internally or as an anesthetic. 4 Some of the diuretics, 
especially potassium acetate, produce an increase in the urinary chlorids. 

In metabolic work it is frequently of advantage to study the effects of 
an ash-free diet upon the pathologic condition. Taylor 5 has, therefore, intro- 
duced such a diet, consisting of the whites of 18 eggs, 120 grams of olive oil 
and 200 grams of crystallized sugar. The work of Goodall and Joslin 6 with 
this diet confirms the earlier views that it is practically impossible to diminish 
the chlorin of the body by more than 10 to 14 per cent, and that the loss 
of water is proportionate to this. 

Estimation of the Chlorids. 

For rough clinical purposes the amount of chlorids in the urine may 
be estimated as follows : A few c.c. of clear, filtered urine, from which albumin 
if present is removed by heating with acetic acid, are placed in a test-tube and 
acidified with 10 drops of chemically pure nitric acid. This mixture is then 
treated with a few drops of 10 per cent, silver nitrate solution. If the chlorids 
are present in normal amount a distinct, curdy white precipitate will settle 
out. If the chlorids be increased a heavy precipitate will be observed, while 
if they be diminished only a cloud without any flakes will be seen. 
Quantitative Determination. 

The best method for such determination is, in the writer's opinion, the 
Arnold modification of Volhard's method. 7 The principle of the test is the 

1 See Barantschik, Deutsch. Arch. f. klin. Med., 1914, CXIV, 167; Landsberg, Ztschr. f. 
Geburtsh. u. Gynak., 1914, LXXVI, 53; Levison, Jour. Am. Med. Assn., 1915, LXTV, 326. 

2 Burnham (Jour. Am. Med. Assn., 1912, LVIII, 851) reports a case of alcoholic cirrhosis 
in which there was a constant decrease in the excretion of the urinary chlorids. 

3 See Forschbach and Weber, Ztschr. f. klin. Med., 1911, LXXIII, 221; also, Meyer, Ibid., 
1912, LXXIV, 352. 

4 See Graham, Jour. Exper. Med., 1915, XXII, 48. 

5 Univ. Cal. Pub., Pathology, 1904, I, 71. 

6 Arch. Int. Med., 1908, 1, 615. 

7 See Bayne- Jones (Arch. Int. Med., 1913, XII, 90) for a simplification of this test; also, 
McLean and Selling, Jour. Am. Med. Assn., 1914, LXII, 1081; Blasucci, Ibid., 1399; Wunder, 
Munch. Med. Wchnschr., 1914, LXI, 2436; McLean and Van Slyke, Jour. Biol. Chem., 1915, 
XXI, 361. 



196 DIAGNOSTIC METHODS. 

precipitation of the chlorids in a definite amount of urine by a standard 
solution of silver nitrate in the presence of an excess of free nitric acid. If 
the precipitate of silver chlorid be filtered from the solution, the excess of 
silver nitrate may be determined in the filtrate by titration with a standard 
solution of potassium sulphocyanate, using a strong solution of iron-ammo- 
nium-alum as an indicator. The urine should be as fresh as possible and 
should contain no nitrites. Albumin, unless present in very large amount, 
need not be removed. It is wise, however, in case the urine shows a high 
albumin content, to acidify the urine with acetic acid, boil, and filter off the 
precipitated albumin. In doing this one should take a definite voLume of 
urine, precipitate as above, and wash the precipitate thoroughly with water 
in order to dissolve any chlorids which may have been retained by the al- 
bumin. The filtrate is made up to a definite volume, which represents the 
amount of urine originally taken. Thus, 20 c.c. of urine are treated as above 
and washed with sufficient water to make 50 c.c. In the test as outlined later, 
in which 10 c.c. of urine are used, 25 c.c. of this filtered albumin-free urine 
will represent 10 c.c. of original urine. 1 

Solutions Necessary. 

(1). A solution of silver nitrate of such a strength that 1 c.c. is equiva- 
lent to 0.01 gram of NaCl or 0.00606 gram of CI. In making this solution 
29.055 grams of pure anhydrous crystallized silver nitrate are dissolved in 
1 liter of distilled water. The chemically pure AgN0 3 as found on the 
market is perfectly reliable and needs only an accurate chemical balance for 
weighing the exact amount. It is essential that this solution should contain 
exactly the amount specified, as the accuracy of the method depends upon 
the correct strength of the volumetric solutions. 

(2). A solution of potassium sulphocyanate of such a strength that 
20 c.c. will correspond exactly to 10 c.c. of the silver solution or, in other words 
so that 2 c.c. of the cyanate solution re necessary to precipitate the silver, 
from each c.c. of the silver nitrate solution. Other workers use solutions 
of potassium sulphocyanate of somewhat different strength, but the simplicity 
of the calculations necessary to determine the chlorids of the urine is much 
increased by such a relation between the two volumetric solutions As po- 
tassium sulphocyanate is very hygroscopic, it is impossible accurately to 
weigh the exact amount necessary to make this solution. We, therefore, 
dissolve a slight excess (9 grams) of potassium sulphocyanate in approximately 
1 liter of water. In order to make this solution correspond exactly to the 
silver solution it is necessary to find out how much water must be added to 
make 20 c.c. of this neutralize 10 c.c. of the silver solution. The technic 
is as follows: ten c.c. of the known solution of silver nitrate are measured 
from a buret and diluted with 50 or 60 c.c. of distilled water. Five c.c. 
of chemically pure nitric acid (specific gravity 1.2) and 5 c.c. of a strong solu- 
tion of iron-ammonium-alum are added and thoroughly mixed. This mixture 
is then titrated with the potassium sulphocyanate solution whose strength. 

1 If the urine be highly colored, it may be cleared with blood charcoal (see Larsson, Biochem. 
Ztschr., 1913, XLIX, 479). 



THE URINE. 197 

is to be determined. The principle of this titration is that the KCNS first 
combines with the AgN0 3 , forming a white precipitate of silver sulphocyanate. 
At the exact point at which this combination is complete the potassium sulpho- 
cyanate will combine with the iron of the indicator forming sulphocyanate 
of iron which is distinctly red in color. The titration is, therefore, carried 
to the point at which a permanent faintly reddish-brown color appears on 
shaking the mixture. The number of c.c. of the sulphocyanate solution neces- 
sary to produce this end point is then read off from the buret and we are 
ready for our correction. As the sulphocyanate solution was intentionally 
made too strong, the titration should yield fewer than 20 c.c. of this solution. 
Supposing 18.5 c.c. of sulphocyanate solution were used we must obviously 
add to every 18.5 c.c. of the remaining sulphocyanate solution 1.5 c.c. of 
water according to the equation C = — ^ in which C represents the number 
of c.c. of water which must be added to the remaining solution; N the total 
number of c.c. remaining after titration; n the number of c.c. consumed 
in one titration, and d the difference between the number of cubic centimeters 
theoretically required and that actually used in one titration. 

The calculation would, therefore, run as follows: 

C= 98l '| > l I ' 5 = 79.58. We must, therefore, add to the remaining 981.5 
c.c. of potassium sulphocyanate solution 79.58 c.c. of water to make the sulpho- 
cyanate solution of such a strength that 20 c.c. will exactly precipitate the 
silver from 10 c.c. of the AgN0 3 solution. 

(3). A cold saturated solution of iron-ammonium-alum. This must 
be absolutely chlorin free. 

(4). Chemically pure nitric acid, chlorin-free and having practically 
no trace of nitrous acid. Specific gravity 1.2. 

Technic. 

Ten c.c. of urine or 25 c.c. of the diluted urine from which the albumin 
has been removed are accurately measured with a pipet and placed in a 
100 c.c. volumetric flask. Five c.c. of nitric acid, 50 c.c. of water, and 20 
c.c. of the standard silver solution are then added and the mixture thoroughly 
shaken. After this mixture has stood for about 10 minutes distilled water 
is added up to the graduating mark of the flask, after which the whole is thor- 
oughly mixed and the precipitated silver chlorid allowed to settle. This 
mixture is then filtered through a perfectly dry filter into a thoroughly dry 
50 c.c. volumetric flask. This 50 c.c. of filtrate will represent, therefore, 
only 5 c.c. of urine, but the calculation made later will compensate for this. 

This 50 c.c. is then poured into a beaker of about 250 c.c. capacity and 
the volumetric flask is thoroughly washed out with water, the washings being 
added to the solution in the beaker. Five c.c. of the alum solution are then 
added and the mixture titrated with the potassium sulphocyanate solution 
to the appearance of the first permanent reddish tinge of the solution. The 
number of c.c. of sulphocyanate solution, necessary to neutralize the excess 
of silver remaining after the chlorid of silver has been filtered off, is then 
read off from the buret. 



198 DIAGNOSTIC METHODS. 

Calculation. 

As 20 c.c. of the sulphocyanate solution are equivalent to 10 c.c. of the 
silver solution, it is evident that the number of c.c. of silver solution not used 
in the precipitation of the chlorids corresponds to the number of c.c. of sulpho- 
cyanate solution necessary to neutralize the 50 c.c. of the filtrate. We, there- 
fore, subtract the number of c.c. of sulphocyanate solution used from 20 
(the number of c.c. of silver solution added) and obtain directly the number 
of c.c, of silver solution necessary to precipitate the chlorids in 10 c.c. of 
urine. As each c.c. of silver solution represents 0.01 gram of NaCl or 0.00606 
gram of CI, multiply these factors by the number of c.c. used, the percentage 
of chlorids in the urine being obtained by multiplying the amount of chlorids 
in 10 c.c. by 10, and the total amount by simply multiplying this figure by the 
number of hundreds of c.c. in the total 24-hour specimen of urine. 

If the urine is very highly colored it is advisable to add a few drops of 
a concentrated solution of potassium permanganate before the titration. This 
will usually decolorize the urine so that the end point will be much more distinct. 

Purdy's Centrifugal Method. 

This method, while having nothing in common with the accuracy of the 
preceding one, is very convenient and has the advantage of yielding quick 
results which are clinically available. 

Ten c.c. of clear, filtered, albumin-free urine are placed in a centrifuge 
tube which is graduated to 15 c.c. One c.c. of strong nitric acid and 4 c.c. 
of a 5 per cent, solution of silver nitrate are then added. The tube is shaken 
by inversion and the mixture allowed to stand for a few minutes, after which 
it is placed in the centrifuge and whirled for three minutes at the rate of 1,200 
revolutions per minute. The bulk percentage of silver chlorid is then read 
off, from which the percentage by weight both of sodium chlorid and of 
chlorin, equivalent to the precipitated silver chlorid, may be calculated. 
One per cent, by bulk represents 0.13 per cent, by weight of NaCl and 0.08 
per cent, of CI. 

As previously stated, the amount of chlorin in the urine depends upon 
the amount ingested, ranging normally between 10 and 15 grams. By the 
use of Folin's standard diet, which contains 6.2 grams of CI, the excretion 
is found to be 6.1 grams of CI in 24 hours. On the ash-free diet of Taylor 
the excretion at the end of 12 days of such a diet was 0.17 gram of CI in the 
24 hours. 

(2). Phosphates. 

The phosphates occurring in the urine are the sodium, potassium, cal- 
cium, and magnesium salts of the tribasic orthophosphoric acid (H 3 P0 4 ). 
As previously stated, in the discussion on the reaction of the urine, normal, 
clear, acid urine contains no dibasic monacid phosphates, but all of the phos- 
phates under these conditions are of the monobasic diacid type. If the urine 
becomes less acid or amphoteric in reaction we find, however, in addition 
to the above, the disodium monohydrogen phosphate, the monocalcium phos- 
phate, and the monomagnesium phosphate; while if the urine be alkaline 



THE URINE. I99 

we may find the neutral phosphates in the ascendency. It must be remembered, 
therefore, that the normal acidity of the urine is not strictly regulated by 
variations in the relative proportions of the monosodium dihydrogen phosphate 
and of the disodium monohydrogen phosphate as usually stated. Besides 
these mineral phosphates, phosphoric acid is found in the urine in combination 
with glycerin as glycero-phosphoric acid, which is derived largely from the 
hydrolytic cleavage of lecithin compounds. According to Mandel and Oertel, 
Mathison, 1 and Yoshimoto, 2 the output of organically bound phosphorus is 
not increased by a phosphorus-rich diet, while Kondo 3 shows that, although 
absolutely increased in amount, its relation to total P 2 5 is diminished. 

The larger portion of the urinary phosphoric acid is derived from the 
food, the smaller portion coming from the metabolism of the tissue protein, 
especially the nucleins. This endogenous phosphoric acid may be of special 
importance as variations will be found depending upon the degree of destruc- 
tion of the lecithin and nuclein compounds. It is to be remembered here that 
not all of the phosphoric acid ingested is excreted, as between a fourth and 
a third of the total quantity may remain in the feces in combination with 
calcium. In studying the effects of increased ingestion of phosphates, the 
feces must, therefore, be examined quite as closely as the urine. 

Physiologic Excretion. 

The amount of phosphoric acid excreted in the 24 hours is always ex- 
pressed in terms of P 2 5 . The normal P 2 5 excretion of the adult varies 
from 1 to 5 grams with an average of about 3.5 grams. The figures of Folin, 
based upon a diet containing 5.9 grams of P 2 5 , show this excretion to average 
3.87 grams in 24 hours, while patients on an ash-free diet eliminate approxi- 
mately 0.75 gram. In this excretion the phosphates of sodium and potas- 
sium usually exceed those of calcium and magnesium, the former being ex- 
creted in the amounts of 2 to 4 grams in the 24 hours, the latter from 1 to 1.5 
grams. Little data exists regarding pathologic variations in the relation 
of these two types of phosphates so that no conclusion may at present be 
drawn. The excretion of P 2 5 will vary with the food, especially with the 
amount of calcium and magnesium of the food. These bases combine in 
the intestine with the phosphoric radical forming phosphates which are difficultly 
soluble. 4 This fact is taken advantage of by Croftan in the administration 
of calcium salts to precipitate the phosphates and thus diminish their activity 
in conditions attributable to uric acid. 

The phosphates are increased on an animal diet and diminished on a 
vegetable diet as Zulzer has shown. During starvation an increase of the 
phosphates may be observed, as an indication of decomposition of the tissues. 
Administration of phosphates at this time will usually lead to a retention 
to counterbalance the previous loss. This same fact was observed in the 
discussion of the chlorids and may be stated as a general law, that an increased 

1 Biochem. Jour., 1909, IV, 274. 

2 Ztschr. f. physiol. Chem., 1910, LXIV, 464- 

3 Biochem. Ztschr., 1910, XXVIII, 200. See, also, Plimmer, Biochem. Jour., 1913, VII, 43. 

4 See Wurtz, Biochem. Ztschr., 1912 XLVI, 103; also, Knox and Tracy, Am. Jour. Dis. 
Child., 1914, VII, 409. 



200 DIAGNOSTIC METHODS. 

excretion is followed by a retention and a retention by an increased elimination. 
It must be stated, however, that an insufficient supply of phosphoric acid 
is not compensated for by such a great retention of phosphates as of the chlor- 
ids. The organism eliminates even more phosphoric acid in starvation than 
in cases of deprivation of salt, as the decomposing protein sets free the salts 
bound up with it. Many attempts have been made to determine where and 
in what form phosphoric acid is retained in the body and where and from 
what sources the body draws upon it for excretion. It is a difficult matter 
to determine what amount of the phosphoric acid retained reaches the bones, 
what portion is devoted to the soft tissues, and how much of it remains organ- 
ically combined in the body (Magnus-Levy). It has been found that the 
relation between the excretion of phosphoric acid and nitrogen is normally 
about one to seven, the same relations which exist between the amount of 
nitrogen and phosphoric acid in the human muscular tissue. It is, therefore, 
plausible to assume that a retention of both nitrogen and phosphoric acid will 
lead to a deposition of increased flesh. In starvation we find that this relation 
is markedly disturbed, the phosphoric acid being both relatively and absolutely 
increased. Such being the case the loss of P 2 5 must be largely sustained 
by the bones, which are relatively poor in nitrogen. For a full discussion 
of this subject the writer would refer to von Noorden's work on Metabo- 
lism and Practical Medicine, which gives great detail regarding all phases of 
metabolism. 

The phosphates are increased during hard muscular exercise, while mental 
exercise seems to lead to a diminished excretion of the alkaline phosphates 
and an increased output of the earthy phosphates. The ingestion of large 
quantities of water is frequently associated with an increased elimination 
of the phosphates, although this is later followed by a slight retention. 

It not infrequently happens that a freshly voided urine shows a marked 
turbidity and even precipitation due to the deposit of earthy phosphates. 
This has been supposed to be due to an increased output of the phosphates, 
but it is now known to be nothing but the natural consequence of a change 
of urinary reaction from acid to alkaline. This condition which has been 
called " phosphaturia" would, therefore, much more appropriately be styled 
" alkalinuria" This subject will be discussed in a later section to which 
the reader is referred. 1 

Pathologic Variations. 
A diminished elimination may be observed in cases of acute febrile 
disease, especially at the height of pneumonia. The degree of diminution 
is usually proportionate to the severity of the disease and usually lessens as 
convalescence comes on. According to Gouraud, the earthy phosphates are 
considerably reduced in pneumonia, while in tuberculous conditions the 
phosphates are increased, an interesting point in differential diagnosis. This 
retention in pneumonia as well as in the other acute febrile diseases is pos- 
sibly due to the renal insufficiency which may be very great in such conditions. 

1 See Diinner, Deutsch. Med. Wchnschr., 1915, XLI, 973. 



THE URINE. 20I 

This diminished phosphatic excretion may not always obtain in the acute 
febrile conditions, in some cases a sudden increased output being observed. 
In typhoid fever Robin believes an increased elimination during the febrile 
rise to be an unfavorable sign, while an increase during defervescence indi- 
cates a favorable prognosis. 

The phosphates appear to be diminished in most chronic diseases. In 
all renal diseases, whether acute or chronic, a diminished excretion is present 
due to the renal insufficiency. This diminished phosphoric acid excretion 
is regarded by Purdy 1 as a factor almost as constant as is the excretion of al- 
bumin. In gout the phosphoric acid excretion runs parallel to that of uric 
acid, decreasing immediately preceding the acute attack and rising as the 
attack subsides. In cases of pregnancy a diminished excretion is observed 
which is attributable to the withdrawal of phosphoric acid from the maternal 
organism for the purpose of the fetal bone formation. In certain bone dis- 
eases, such as osteomalacia, a diminished excretion is usually observed, although 
at times an actual increase is seen. The earthy phosphates, especially, are 
diminished in these latter conditions while the alkaline phosphates may be 
increased. In cases of myositis ossificans the excretion of inorganic phosphates 
does not seem to be much affected as one might expect from the new bone 
formation. 

In cases of hystero-epilepsy the phosphates are diminished, the diminu- 
tion usually being proportionate to the intensity of the attack, while in true 
epilepsy the phosphates appear to be more or less markedly increased. It 
is in just the nervous diseases that one would expect to find much variation 
in the phosphatic excretion, but very few data are found bearing on this sub- 
ject. Folin and Shaffer find that in the periods of nervous excitement the 
relative amount of phosphoric acid is diminished, but that the absolute amount 
is little changed. 

In Addison's disease, hepatic cirrhosis, acute yellow atrophy, and chronic 
lead-poisoning we may find an extensive decrease of phosphates in the urine. 

In certain cases which show most of the symptoms of diabetes mellitus 
without any sugar output, a phosphatic increase is observed in the urine. 
This condition has been called "phosphatic diabetes" and may be associated 
with the excretion of as high as 10 grams of P 2 5 within 24 hours. In true 
diabetes mellitus the phosphates may be increased at one time and diminished 
at another, as there seems to be an inverse ratio between the excretion of 
sugar and that of the phosphates. 

The phosphates seem to be increased in cases of pseudoleukemia, leuke- 
mia, hemorrhagic purpura, in cases of acute or chronic inflammatory 
processes of the genito- urinary tract, and in cyclic vomiting of children. 

As previously stated, the output of urinary nitrogen bears a relation of 
about seven to one to that of the phosphate excretion. This relation has 
been termed the "relative value" of phosphoric acid and represents the amount 
of P 2 5 corresponding to 100 grams of N. Normally this ranges between 
15 and 20. 

Practical Urinalysis, Phila., 1900, p. 56. 



202 DIAGNOSTIC METHODS. 

Estimation of Phosphates. 

Ten c.c. of urine are rendered alkaline with ammonia. The earthy phos- 
phates are precipitated in the form of a flocculent precipitate and may be 
roughly estimated by the volume of the precipitate. 

If this alkalinized urine be filtered and the filtrate acidified with acetic 
acid, the addition of a few drops of ferric chlorid or of uranium nitrate solu- 
tion will precipitate the alkaline phosphates. 

These methods are purely qualitative and can have no clinical value 
beyond giving a general idea of the relative amounts of the earthy and alkaline 
phosphates. Instead of ferric chlorid or uranium solution, magnesium 
mixture may be used for this purpose. 

Quantitative Determination. 

The best method for the estimation of the urinary phosphates is that 
of titration with uranium nitrate or acetate solution. The principle of this 
method is that phosphoric acid compounds in acetic acid solution give, on 
treatment with uranium nitrate, a yellowish-white flocculent insoluble pre- 
cipitate of uranium phosphate (U0 2 HP0 4 ). As a means of recognizing the 
point at which an excess of uranium solution is present in the titrated fluid, 
one may use either a solution of ferrocyanid of potassium which gives a dis- 
tinct brownish color at the end point, or, preferably, a few drops of tincture 
of cochineal, which gives a grass-green color and has the advantage that it 
can be added directly to the titrated fluid, which is not the case with the ferro- 
cyanid of potassium. 

Necessary Solutions. 

(i). A solution of uranium nitrate or acetate of such a strength that 20 c.c. 
shall correspond to 0.1 gram of P 2 O s . It is a matter of absolute indifference 
whether the acetate or the nitrate be used, but the writer prefers the nitrate 
as this is more easily obtained in the pure state. In making up a solution 
of uranium nitrate of the above strength, one may not rely implicitly on the 
weighing, as the uranium nitrate may contain impurities or excess water 
and thus vitiate the results. It is, therefore, necessary to have a standard 
phosphate solution against which the uranium solution may be titrated. 

The usual solution recommended by various writers is one of disodium 
monohydrogen phosphate. This salt varies in its degree of hydration and 
its solutions do not keep well. Moreover, it is absolutely necessary when this 
salt be used that a definite amount of it be taken and converted into sodium 
pyrophosphate, after which a corresponding dilution of the solution must be 
made to make it of such a titer that every 50 c.c. shall be equivalent to 0.1 
gram of P 2 5 . In view of these facts, the writer is accustomed to follow 
the suggestion of Giles 1 and use chemically pure dihydrogen monopotassium 
phosphate. This salt crystallizes well without any water of crystallization 
and does not alter on exposure to the air. 

This solution is to be made such a strength that 50 c.c. corresponds to 
0.1 gram of P 2 5 , in other words a liter must contain 2 grams of P 2 O s . In 
1 Sutton's Volumetric Analysis, Philadelphia, 1904, p. 294. 



THE URINE. 



203 



order to find out just how much of this salt must be dissolved in a liter of 
water we must have recourse to a simple calculation. The formula of dihydro- 
gen monopotassium .phosphate is KELjPC^, its molecular weight being 136. 
Two molecules of this salt are necessary to yield one molecule of P 2 O s accord- 
ing to the equation 

2 KH 2 P0 4 =P 2 O s + K 2 + 2H 2 0. 

If, therefore, one liter of the solution must contain 2 grams of P 2 O s , the amount 
of KELjPC^ which must be dissolved in a liter is easily calculated from the 
following proportion : 

272 : 142 :: x : 2. x = 3.83. 

We, therefore, dissolve 3.83 grams of dihydrogen monopotassium phosphate 
in 1 liter of water and obtain directly a solution which contains 2 grams of 
P 2 O s or one in which every 50 c.c. is equivalent to 0.1 gram of P 2 O s . It is 




Fig. 71. — Volumetric flasks. 

perhaps, needless to add that this solution should be made in an accurately 
standardized volumetric flask, and at the temperature at which the flask 
is calibrated. 

Having thus obtained our standard phosphate solution, we are now in 
a position to make up our standard uranium nitrate solution, the titer of which 
must be such that 20 c.c. corresponds to 0.1 gram of P 2 O s , or, in other words, 
one liter of which must be equivalent to 5 grams of P 2 O s . The formula of 
uranium nitrate is U0 2 (N0 3 ) 2 6H 2 0, its molecular weight being 502.6. Ura- 
nium nitrate combines with dihydrogen potassium phosphate according to the 
following equation : 

U0 2 (N0 3 ) 2 + KH 2 P0 4 =U0 2 HP0 4 + HN0 3 + KN0 3 . 



204 DIAGNOSTIC METHODS. 

As seen above, two molecules of the dihydrogen phosphate are necessary to 
yield one molecule of P 2 5 . We will, therefore, have, when uranium nitrate 
acts upon the dihydrogen phosphate, only the equivalent of 1/2 molecule 
of P 2 5 ; that is, 71 parts. As the uranium solution must contain 5 grams of 
P 2 O s to the liter we may then calculate how much uranium nitrate is necessary 
to form the equivalent of such a solution by the following proportion: 

502.6 : 71 :: x 15. x = 35.3Q. 

Were we absolutely certain of the purity and state of hydration of our uranium 
nitrate, all that would be necessary would be to weigh out this exact amount. 
As this is not the case, we weigh out a slight excess (35.75 grams) and dissolve 
in one liter of distilled water. We are now ready to determine the strength 
of the uranium solution as follows: 

Fifty c.c. of the dihydrogen monopotassium phosphate solution are placed 
in a beaker and treated with a few drops of tincture of cochineal and 5 c.c. 
of acetic acid mixture (see below, solution 2). Some workers prefer the ad- 
dition of potassium ferrocyanid as an indicator, but this does not give as distinct 
a contrast at the end point, and if tests are made by adding a drop of the 
mixture to the ferrocyanid solution on a white plate, loss of substance must 
occur. This mixture is then heated and titrated, as soon as the boiling point 
is reached, with the uranium solution until a trace of a distinct green color 
becomes permanent on stirring the mixture. Duplicate determinations are 
then made, the results of which should agree exactly with the original. The 
number of c.c. of uranium solution used is then read off and we are prepared 
for the calculation of the amount of water which must be added to stand- 
ardize the solution. 

As 20 c.c. of this uranium solution should correspond exactly to 50 c.c. 
of the standard phosphate solution, we may, for the sake of example, use the 
same figures given for obtaining the dilution in the case of the sulphocyanate 
solution discussed under the heading of Chlorids. Thus if 18.5 c.c. of ura- 
nium solution were used we must add, according to the previous explanation, 
79.58 c.c. of distilled water to the remaining 981.5 c.c. of uranium solution 
in order to make every 20 c.c. equivalent to 0.1 gram of P 2 O s . 

(2). An acetic acid mixture prepared by dissolving 100 grams of sodium 
acetate and 30 grams of glacial acetic acid in sufficient water to make 1000 c.c. 
This solution must be added in the determination of the urinary phosphates 
in order to overcome the influence of the nitric acid liberated in the reaction 
and to convert any monacid phosphates into the diacid type. 

(3). An indicator, preferably tincture of cochineal prepared by digesting 
the ground cochineal bugs in 25 per cent, alcohol and filtering. This indicator 
has the advantage that it may be added directly to the solution to be titrated, 
while potassium ferrocyanid must be used by the plate method of adding 
a few drops of the solution to the indicator after each addition of uranium 
solution. 

Technic. 

Fifty c.c. of clear filtered urine are placed in an Erlenmeyer flask and 



THE URINE. 205 

treated with 5 c.c. of the acetic acid mixture, for the purpose of transforming 
any monacid phosphates into the diacid form and of neutralizing the nitric 
acid formed during the titration. A few drops (5 to 10) of tincture of cochineal 
are added, the mixture heated to the boiling-point, and then titrated as de- 
scribed above. It is wise invariably to run duplicate determinations. After 
each addition of the uranium nitrate the precipitate is allowed to settle so 
that one may see more clearly the first trace of any green coloration or 
precipitate. 

The calculation is as follows: Supposing 10 c.c. of the uranium solution 
were used, the corresponding amount of P 2 5 in the 50 c.c. of urine examined 
would then be found from the equation: 

20 : 0.1 : : 10 : x. x=o.o5. 
The percentage of P 2 5 would, therefore, be 0.1 (2 x 0.05). If the total 
24-hour urine were 1,500 c.c, the total P 2 5 excretion would be obviously 
1.5 grams. 

Total Phosphoric Acid. 

The above determination gives the total P 2 5 excretion as far as the 
inorganic phosphates are concerned, but it does not take into consideration 
the organically bound phosphorus. This is usually very small in amount, 
but it is sometimes of value to know whether any variations exist. 

This may be determined by incinerating 50 c.c. of urine and determin- 
ing the total phosphoric acid in the ash. This, however, is not as easily done 
as might be imagined, owing to the fact that the urinary residue is difficultly 
incinerated. The writer prefers, therefore, the method of Asboth-Folin 
which will be discussed in the section on Sulphates. The mass fused by 
this method is dissolved in hot water, the solution filtered and washed with 
hot water. This solution is then slightly acidified with HC1, treated with the 
acetic acid mixture, and titrated as in the previous determination. The 
difference between the figures obtained here and those obtained by direct 
titration of the urine will yield the amount of organically-bound phorphorus 
in terms of P 2 5 . The normal output of this organic phosphorus varies from 
0.01 to o. 1 gram in 24 hours, forming about 2 or 3 per cent, of the total P 2 5 . 
Phosphorus-rich diets increase the absolute amount but diminish the relative 
percentage. 

Purdy's Centrifugal Method. 

This method cannot be relied upon for accurate results in metabolic 
work, but may be of some service from the clinical standpoint. Ten c.c. 
of clear filtered urine are placed in a centrifuge tube graduated to 15 c.c. Two 
c.c. of 50 per cent, acetic acid, and 3 c.c. of 5 per cent, uranium nitrate solu- 
tion are then added and thoroughly mixed with the urine by inversion of the 
tube. The tube is then placed in the centrifuge and operated at a speed of 
1,200 revolutions for three minutes. According to Purdy, 1 per cent, by 
bulk of uranium phosphate equals 0.04 gram of P 2 5 in each 100 c.c. of 
urine. Each succeeding percentage by bulk increases by the figure 0.0 1. 
Thus a bulk percentage of five of uranium phosphate would equal 0.04 plus 



206 DIAGNOSTIC METHODS. 

0.04, or 0.08 gram of P 2 5 in each 100 ex. These figures are much at vari- 
ance with those of Ogden, who states that "he has found that each 1/10 of 
a c.c. of precipitate calculated as P 2 5 is equivalent to 0.0225 P er cent, by 
weight." Owing to these differences, the writer would suggest that the bulk 
percentage be stated as such rather than as parts by weight of P 2 8 . 

(3). Sulphur Compounds. 

The sulphur is present in the urine in three forms: (1) preformed or neutral 
sulphates; (2) ethereal or conjugated sulphates, sulphuric acid in combination 
with aromatic compounds, and (3) neutral, unoxidized, or organic sulphur. 
The total output of sulphur depends essentially upon the protein metabolism, 
both of that of the tissues and of the food. 1 It is to be remembered that the 
sulphur elimination is much less accurate than that of the nitrogen as an indi- 
cation of the degree of protein metabolism, owing to the fact that different 
protein substances vary in their sulphur-content. The daily excretion of 
sulphur, in terms of S0 3 , varies from 1 to 3.5 grams, when the subject is upon 
a mixed diet. Ordinarily, the ethereal sulphates form about one-tenth of the 
total output. The neutral sulphur does not vary under normal conditions 
as far as its absolute amount is concerned, but we note, on changing the 
diet to one which is relatively free in protein material, that the relative amount 
of the neutral sulphur is markedly increased. Thus, Folin finds on a diet 
containing 18.9 grams of nitrogen and 3.8 grams of S0 3 the daily excretion 
of total S0 3 is 3.31 grams, of which the inorganic S0 3 is 2.92 (87.8 per cent, 
of total), the ethereal S0 3 is 0.22 (6.8 per cent.), and the neutral S0 3 0.17 
(5.1 per cent.). On a nitrogen-free diet, consisting of cream and arrowroot, 
a total S0 3 excretion of 1.04 grams is noted, of which 0.63 gram (60.6 per 
cent.) is traceable to the inorganic S0 3 , 0.12 (11.5 per cent.) to the ethereal 
S0 3 , and 0.29 (27.9 per cent.) to the neutral S0 3 . We are, therefore, con- 
fronted with the following fact, "the distribution of the sulphur in urine among 
the three chief normal representatives, inorganic sulphates, ethereal sulphates, 
and 'neutral sulphur,' depends on the absolute amount of sulphur present." 

On the ash-free diet of Taylor we find, according to Goodall and Joslin, 
a total S0 3 excretion of 0.96 gram, of which the inorganic S0 3 forms 0.71 
gram (74 per cent.), the ethereal sulphates 0.05 gram (5.2 per cent.), and 
the neutral sulphur 0.2 gram (20.8 per cent.). 

It is doubtless true that practically all of the urinary sulphur is derived 
from protein metabolism, a definite relation being usually established between the 
nitrogenous and sulphur output. Normally, N : S0 3 1:5:1, apparently 
regardless of whether the patient is on a nitrogen-rich or a nitrogen-free diet; 
the absolute amounts of each, however, differ markedly, depending upon the 
diet. Folin's figures show that as the total urinary sulphur is reduced, the 
percentage represented by the inorganic sulphates sinks from about 90 to 
60 per cent. This fact has been expressed in the above quotation from 
Folin. 

The reduction in the inorganic sulphates must be made up by a relative 

1 See Cathcart and Green, Biochem. Jour., 19 13, VII, 1. 



THE URINE. 207 

increase in the other forms of sulphur. The ethereal sulphates have for 
a long time been held to be an accurate index of the degree of absorption 
of the products of intestinal protein decomposition. There can be no question 
but that increased intestinal decomposition is associated with increased out- 
put of the conjugated sulphuric acids, especially the indoxyl and skatoxyl 
sulphuric acids. These ethereal sulphates are diminished on a milk diet 
or on the cream and arrowroot diet of Folin or the vegetarian diet adopted 
by many of Chittenden's subjects. This is true especially as regards the 
urinary indican which is absolutely negative in such cases, while the total 
amount of ethereal sulphate is diminished only about 50 per cent. Such 
being the case we must have some other than intestinal origin for the large 
relative increase of the ethereal sulphates under a nitrogen-free diet. I 
quote from Folin: 1 "(1) The urinary indican is not to any extent a product 
of the general protein metabolism, is therefore probably, as is generally sup- 
posed, a product of intestinal putrefaction, and may consequently be assumed 
to indicate approximately the degree of putrefaction in the intestinal tract. 

(2) The ethereal sulphates can only in part be due to intestinal putrefaction, 
and neither their absolute nor their relative amount can be accepted as an 
index of the extent to which the putrefaction is taking place in the intestines. 

(3) The ethereal sulphates, on the contrary, represent a form of sulphur 
metabolism which becomes more prominent when the food contains little 
or no protein." Here the sulphuric acid is conjugated with aromatic bodies 
formed from decomposition of tissue protein. 

Pathologic Variations. 

The sulphates, as a whole, must be increased in any condition associ- 
ated with increased protein catabolism. Thus we find in febrile conditions 
an increased output of sulphur corresponding to the intensity of the process, 
this increase being followed by a diminution as convalescence comes on. 
An increased elimination of sulphates has been observed in leukemia, diabetes 
mellitus and insipidus, progressive muscular atrophy, and following the use 
of such drugs as morphin, potassium bromid, sodium salicylate and acet- 
anilid. From the clinical standpoint the elimination of sulphates has little 
practical value, the variation in the amounts of ethereal sulphates and neutral 
sulphur being the chief factors of value. 2 

While, as shown above, the ethereal sulphates are subject to great varia- 
tion, the indoxyl-potassium sulphate (indican) varying according to the degree 
of intestinal decomposition, we find some points of clinical interest in their 
study. As the putrefactive processes normally occur below the ileo-cecal 
valve, any condition increasing such decomposition with a consequent in- 
crease of urinary indican would indicate trouble in the lower bowel, more 
frequently of the chronic type. They are increased in cases showing abnormal 
intestinal absorption, as, for instance, in typhoid fever, intestinal tuberculosis, 
peritonitis, and chronic intestinal catarrh. Obstructive jaundice is usually 

1 Loc. cit. 

2 See Stadtmiiller and Rosenbloom, Arch. Int. Med., 1913, XII, 276; Ross, Ibid., 1914, 
XIII, 889; Greenwald, Ibid., 1914, XIV, 374. 



208 DIAGNOSTIC METHODS. 

associated with increase in the output of indican as the bile seems to have 
a great influence upon putrefactive processes in the intestine. They appear 
to be increased in cholera, while in ordinary diarrhea they are diminished 
in absolute amount, but may be relatively increased. In cases of gastric 
hypoacidity associated with bacterial decomposition in the stomach the urine 
may show a marked increase of indican. In acute nephritis a very intense 
indican reaction may be obtained, while in the chronic form the amount of 
indican is usually diminished. In cases of pus-formation almost anywhere 
within the system an increased elimination of ethereal sulphates may be ob- 
served due to the absorption of the products of decomposition of the pus. 
This point is of some importance in differentiating purulent from non-purulent 
affections of various organs. The writer has seen a persistent intense indi- 
canuria, associated with middle-ear infection, which cleared up completely 
after thorough drainage. 

Neutral Sulphur. 

The neutral sulphur does not have a very definite relation to the amount 
of sulphur of the intake or to the amount formed in the decomposition of 
tissue protein. While it is said to vary in amounts representing from 12 
to 15 per cent, of the total S0 3 of the urine, this must be true only when the 
absolute amount of excreted S0 3 is taken into consideration. By this is meant 
that although the absolute amount of neutral sulphur does not normally vary 
to any great extent, its percentage relation to the total sulphur varies with 
the amount of sulphur. Thus Folin finds on an intake of 3.8 grams of S0 3 
an output of 0.17 gram of neutral S0 3 , while on a nitrogen-free diet the out- 
put is 0.2. 

The nature of the neutral sulphur of the urine is somewhat uncertain, 
as we seem to have only two well-established bodies, namely, the sulpho- 
cyanates and hydrogen sulphid. The sulphocyanates are derived largely 
from the absorption of material from the saliva and represent approximately 
one-third of the total neutral sulphur. The hydrogen sulphid may be regarded 
as a decomposition product. Besides these we find cystein, which is an in- 
termediary product of normal protein metabolism, and tauro-carbamic acid, 
which is derived from the biliary material. In cases of jaundice we may find 
as high as 60 per Cent, of the sulphur in the neutral form, which may be due 
to the absorption of this and other biliary material. Traces of chondroitin- 
sulphuric acid, oxyproteic acid, alloxyproteic acid, and uroferric acid con- 
tribute to the neutral sulphur-content of the urine. 

The greatest increase of neutral sulphur is probably associated with 
the presence of cystin which is not normally present in the urine. This is 
undoubtedly derived from abnormal protein decomposition. The sulphur 
in the neutral form may reach as high as 30 or 40 per cent, of the total, due 
to the presence of cystin. The writer will refer to a later section for a discus- 
sion of the subject of cystinuria. 

The variations in the neutral 1 sulphur of the urine must be regarded 
as indicative of abnormal metabolic processes which are not associated with 



THE URINE. 209 

variations in the other types of urinary sulphur. As Folin has shown, "the 
neutral sulphur is not at all due to processes identical or similar to those which 
give rise to indican. The neutral sulphur represents products which in the 
main are independent of the total amount of sulphur eliminated or of pro- 
tein catabolized." 

Estimation of Total Sulphur (A). 
The method followed by the writer in determining the total urinary 
sulphur in terms of S0 3 is the Folin 1 modification of the Asboth-Modrakowsky 
method. Twenty-five c.c. of urine (50 c.c. if very dilute) are measured into a 
large nickel crucible of about 200 c.c. capacity and treated with 3 grams of so- 
dium peroxid. 2 The mixture is evaporated to a syrupy consistency and is then 
carefully heated to dryness. The latter part of this procedure requires about 
15 minutes. The crucible is removed from the flame and allowed to cool. 
Moisten the residue with 1 or 2 c.c. of water, add about 7 grams of sodium 
peroxid and heat the mixture to complete fusion for about 10 minutes. Allow 
the fused mass to cool, add about 100 c.c. of water and heat to boiling for at 
least one-half hour to dissolve the alkali and decompose the peroxid. This 
mixture is transferred to an Erlenmeyer flask (about 400 c.c. capacity) by means 
of hot water and is diluted to about 250 c.c. Concentrated HC1 is slowly added 
to the almost boiling solution until the oxid of nickel (formed in the process) 
just dissolves. This will require about 18 c.c. of acid for 8 grams of Na 2 2 . 
After boiling for a few minutes, the solution should be perfectly clear. If it is 
not clear too much water or too little peroxid were used in the last fusion. 
Allow the mixture to cool and filter from the insoluble residue, if there is any. 
To this clear acid solution add 5 c.c. of very dilute (1 to 4) alcohol and boil for 
a few minutes to remove the last traces of chlorine, which are formed on acidi- 
fying the solution. Now add, drop by drop, 10 c.c. of a 10 per cent, barium 
chlorid solution and allow the precipitate of BaS0 4 to settle out for two days in 
the cold. At the end of this time the precipitate is filtered off, using either ash- 
free filters or, preferably, weighed porcelain Gooch crucibles with properly 
prepared asbestos mats. Wash the precipitate with about 250 c.c. of cold water, 
dry and ignite for 15 minutes. In this heating of the Gooch crucible, the 
flame must touch neither its perforated bottom nor its sides. If the crucible 
be placed upright upon the cover of a platinum crucible, which rests upon a 
platinum triangle, the flame may be applied directly to the bottom of the 
platinum. After the heating, the crucible is placed in the desiccator, is allowed 
to cool and is weighed. Knowing the weight of the crucible and mat before 
and after this process, a simple calculation yields the amount of BaS0 4 obtained 
from the urine taken. If the filter-paper be used, the precipitate is washed as 
previously mentioned. Transfer the filter-paper and precipitate directly to a 
weighed platinum or porcelain crucible, add 3 or 4 c.c. of alcohol and ignite, 
to dry and partially burn the filter-paper. Heat the residue until complete 

1 Jour. Biol. Chem., 1906, I, 131; Jour. Amer. Chem. Soc, 1908, XXXI, 284. See, also, 
Raiziss and Dubin, Jour. Biol. Chem., 1914, XVIII, 297. 

2 Benedict, Denis, and Schmidt use, as the oxidizing agent, a solution of copper nitrate 
and potassium chlorate. 

14 



2IO DIAGNOSTIC METHODS. 

incineration occurs and the ash becomes colorless. Cool the crucible in a 
desiccator and weigh. If the amount of BaS0 4 obtained by either of these 
methods be multiplied by 0.3429, the result is the amount of S0 3 in the urine 
taken. A simple calculation yields the percentage and total amount excreted. 
Determination of Total Sulphates (B). 
Folin's Methods. 
Under this head we determine the neutral (preformed or inorganic) and 
ethereal sulphates. Folin 1 has advanced two methods, the first of which 
is preferable. 

1. Precipitation in the Cold. — 25 c.c. of urine and 20 c.c. of dilute hydro- 
chloric acid (1 to 4), or 50 c.c. of urine and 4 c.c. of concentrated HC1, are 
gently boiled in an Erlenmeyer flask (capacity about 200 c.c.) for 20 to 30 
minutes (never less than 20). To prevent loss of material it is wise to keep 
the flask covered with a small watch-glass during the boiling. The flask 
is cooled for two or three minutes in running water and the contents are then 
diluted with cold water to about 150 c.c. To this solution are then added 10 c.c. 
of a 5 per cent, barium chlorid solution, care being taken not to shake or stir the 
contents during the addition. At the end of an hour or more, the mixture is 
shaken up and filtered. The rest of the determination is as outlined above. 

2. Precipitation in the Heat. — The boiling of the urine with hydrochloric 
acid is conducted exactly as in the preceding method. At the end of 20 to 
30 minutes, the boiling urine is diluted to about 150 c.c. with hot water. The 
mixture is heated once more to the boiling point, is removed from the flame, 
and immediately precipitated by the addition of 5 c.c. of 10 per cent, barium 
chlorid solution, which must be added drop by drop. The mixture is allowed to 
stand for 2 hours in order to cool. The remainder of the process is as pre- 
viously given. 

Determination of Inorganic (Neutral) Sulphates (C). 

About 100 c.c. of water (not less), 10 c.c. of dilute hydrochloric acid 
(1 to 4), and 25 c.c. of urine (50 c.c. if dilute, when a correspondingly smaller 
amount of water is taken) are measured into an Erlenmeyer flask of about 200 
c.c. capacity. 10 c.c. of a 5 per cent, solution of barium chlorid are added, 
using the drop method, the urine solution not being shaken or disturbed during 
this addition. At the end of an hour or more, the mixture is shaken and filtered. 
The remaining steps are given above. The figure obtained here represents the 
amount of S0 3 referable to the preformed sulphates. 

The ethereal sulphates (D) need not be determined separately as the 
difference between the total sulphates (B) and the inorganic sulphates (C) 
will represent the amount of these ethereal sulphates in the specimen examined. 
If the S0 3 referable to the total sulphates (B) be subtracted from the S0 3 of the 
total sulphur (A) the remainder represents the neutral sulphur in terms of S0 3 . 

Purdy's Centrifugal Method. 

Ten c.c. of clear urine are placed in a centrifuge tube and to it are added 
5 c.c. of barium chlorid mixture, consisting of four parts of barium chlorid, 

1 Loc. cit. See, also, Rosenheim, Biochem. Jour., 1914, VIII, 143. 



THE URINE. 211 

one part of concentrated hydrochloric acid, and 16 parts of distilled water. 
The tube is inverted to insure mixing of the reagent and urine and is allowed 
to stand for a few minutes. It is then placed in a centrifuge and whirled 
three minutes at the rate of 1,200 revolutions per minute. Each percentage 
of BaS0 4 by bulk represents approximately 0.25 per cent, of S0 3 by weight. 
This result can, of course, not be as accurate as the preceding, but has some 
clinical advantage. 

(4). Carbonates. 

A freshly voided specimen of urine may contain small quantities of car- 
bonates and bicarbonates and some free carbonic acid. The amount of 
free carbonic acid varies with the degree of acidity of the urine and the amount 
of carbonate-forming material in the food. It has been found that vegetable 
foods are almost always productive of an alkaline urine, owing to the fact 
that the organic acids of the vegetables may be converted into carbonates 
and excreted as such. The carbonate which most frequently forms as a sedi- 
ment in the urine is calcium carbonate which will be treated in a later section. 

If the urine be acidified and a stream of air passed through this acidified 
urine into a vessel containing a solution of barium hydrate, the carbon-dioxid 
liberated by the acids will unite with the barium forming barium carbonate 
which may be filtered, dried, and weighed. 

(5). Sodium and Potassium. 

These metals exist in the urine in the form of the oxids Na 2 and K 2 0, 
the former being present in amounts of 4 to 7.5 grams, the latter varying 
from 2 to 4 grams. The normal relation between the excretion of these prod- 
ucts is as 5 to 3. 

The sodium is largely derived from the addition of sodium chlorid to 
the diet, while the potassium is a constituent of most vegetable foods. We 
find, therefore, that both of these substances depend largely upon the diet 
in normal cases, while in pathologic conditions the potassium may be excreted 
in larger amounts. 1 Thus we find in fever the potassium salts predominate 
over the sodium compounds up to the time of crisis, after which the sodium 
salts again assume their normal proportions. Increased exercise as well as 
increased decomposition of protein from pathologic causes will tend to increase 
the potassium of the urine. From the clinical standpoint the quantitative 
determination of the sodium and potassium of the urine has little significance. 

(6). Calcium and Magnesium. 

Both of these alkali-earth metals are excreted in the urine largely in 
the form of the phosphates. Calculated as the oxids, the calcium excretion 
(CaO) varies from 0.1 to 0.3 gram and the magnesium (MgO) from 0.15 
to 0.4 gram in 24 hours, the relation between the calcium and magnesium 
excretion being about 1 to 1.5. 

The chief source of these compounds is the food, but it must be remem- 
bered that only a small portion of these substances is excreted in the urine. 

1 See Blumenfeldt, Inaug. Dissert., Berlin, 1912; also, Ztschr. f. exp. Path. u. Therap., 
i9i3,XII, 523. 



212 DIAGNOSTIC METHODS. 

Calcium and, to a less extent, magnesium form compounds with phosphoric 
acid in the bowel and are excreted without being absorbed. Even if these 
compounds be injected subcutaneously the excretion is largely into the intes- 
tine. We should, therefore, study much more closely the calcium metabolism 
by examination of both the feces and urine. 

Little is known regarding the output of either of these metals, but what 
work has been done is more closely connected with the calcium excretion 
than that of magnesium. During starvation calcium oxid is increased both 
relatively and absolutely, its probable source being the bones. It is increased 
to some extent by exercise and is diminished after the administration of alkalies. 
In chronic diseases we note an increase due, probably, to inanition. In tuber- 
cular conditions the calcium output is occasionally found to be diminished, 
while some cases of a marked increase have been noted. In diabetes mellitus, 
as in other conditions associated with an acidosis, the output of CaO may 
be greatly increased, thus running parallel to the ammonia output. In a 
case of myositis ossificans studied by the writer there seemed to be no great 
variation in the calcium output, while the magnesium content of the urine 
was markedly reduced. Austin has reported similar findings. McCrudden 
and Fales report (Arch. Int. Med., 1912, DC, 273) a loss of calcium and a 
retention of magnesium in non-puerperal osteomalacia. The same authors 
(Jour. Exper. Med., 1912, XV, 450) find the calcium excretion in the urine, in 
cases of intestinal infantilism, to be almost negligible. Peabody (Jour. Exper. 
Med., 1913, XVII, 71) shows a retention of calcium and a normal excretion of 
magnesium in pneumonia. 1 

Quantitative Determination. 
Calcium. 

McCrudden 2 has recently introduced a method for this determination 
which, in my opinion, yields the best results. If the urine be alkaline it must 
be made neutral or slightly acid with HC1 and be filtered. If turbid or faintly 
acid to litmus, add 10 drops of concentrated HC1. Two hundred c.c. of the 
clear acid urine are treated with 10 c.c. of a 2.5 per cent, oxalic acid solution 
and 8 c.c. of 20 per cent, sodium acetate solution. Allow the mixture to stand 
over night at room temperature or shake vigorously for 10 minutes. Filter off 
the precipitate of calcium oxalate and wash it Cl-free with 0.5 per cent, 
ammonium oxalate solution. Save the filtrate for the determination of mag- 
nesium. The precipitate of calcium oxalate is then dried on the filter-paper 
at ioo° C. and is placed in a weighed platinum crucible and burned over the 
Bunsen flame until the filter-paper is completely ashed. The blast-lamp is 
then applied and the crucible and contents blasted for 15 or 20 minutes. The 
crucible is dried in the desiccator and weighed, the increase in weight repre- 

1 See, also, Voorhoeve, Deutsch. Arch. f. klin. Med., 1913, CXI, 29. For various studies 
of the metabolism of calcium see Diller and Rosenbloom, Am. Jour. Med. Sc, 1914, CXLVIII, 
65; Bergeim, Stewart and Hawk, Jour. Exper. Med., 1914, XX, 218 and 225; Bookman, Am. 
Jour. Dis. Child., 1914, VII, 436; Rosenbloom and Conn, Arch. Int. Med., 1914, XIV, 263; 
Kleinschmidt, Ber] . klin. Wchnscnr., 1915, LII, 29. 

2 Jour. Biol. Chem., 1910, VII, S3; Ibid., 1911, X, 187. See, also, Lyman, Ibid., 1915, 
XXI, 551. 



THE URINE. 213 

senting the calcium oxid (CaO) in the 200 c.c. of urine. A simple calculation 
will yield the amount of CaO in the 24-hour specimen. 
Magnesium. 

The nitrate and washings obtained in the above determination are treated 
with one-third the volume of 25 per cent, ammonium hydrate. A precipitate 
of magnesium ammonium phosphate (NH 4 MgP0 4 ) occurs, which is allowed 
to settle for several hours, is collected on an ash-free filter, is thoroughly washed 
with water containing one-third its volume of ammonia, and is dried in the 
oven at ioo° C. The filter-paper and precipitate are placed in a weighed 
platinum crucible and burned until the filter-paper is completely destroyed, 
after which the crucible and contents are heated over the blast-lamp for 15 
minutes. This heating converts the magnesium ammonium phosphate 
(NH 4 MgP0 4 ) into Magnesium pyrophosphate (Mg 2 P 2 7 ); one part of mag- 
nesium pyrophosphate represents 0.36243 part of MgO. All that is neces- 
sary, therefore, is to multiply the amount of magnesium pyrophosphate 
by the above factor to obtain the amount of MgO in 200 c.c. of urine. 

(7). Iron. 

Iron is practically always present in the urine in organic combination. 
The amount actually present is very small, being given by Magnier as varying 
between 3 and 1 1 mg. to the liter, while Neumann and Mayer find the normal 
output to average 0.983 mg. The urinary iron has little clinical significance. 
It is increased in fever, in malaria, diabetes mellitus, and pernicious anemia, 
the amounts in some cases running as high as 20 mg. during the 24 hours. 

Any method for the estimation of the urinary iron must be very delicate 
and very accurate. The urine must be completely incinerated, the method 
of Neumann 1 employing a mixture of concentrated sulphuric and nitric acid 
being by far the best. 2 

(c). Organic Constituents. 

(1). Nitrogenous Bodies. 
(a). Total Nitrogen. 

From the metabolic standpoint the estimation of the total nitrogen of 
the urine is one of the most important features of its chemical examination. 
The excretion of total nitrogen varies with the amount of nitrogen of the 
food and with the degree of tissue metabolism. Normally, the system so 
adapts itself to the nitrogenous intake that the excretion of nitrogen, in the 
urine, feces, perspiration, etc., is equal to the intake. In other words, a normal 
person is in nitrogenous equilibrium. This subject of nitrogenous equilibrium 
is of such great importance and the factors which influence it so varied that 
the writer feels that it will be unwise to discuss it briefly for fear of not pre- 
senting it clearly to the student's mind. As a full discussion of this subject 
would be too extensive for the scope of this work the writer must be content 
with reference to the admirable discussion by Magnus-Levy 3 in von Noorden's 
Hand-book of Pathology of Metabolism. 

1 Arch. f. Anat. u. Physiol., Physiol. Abth., 1900, S. 159. 

2 See Goodman (Jour. Biol. Chem., 19 12, XII, 37) for the urinary iron in pneumonia. 

3 Chicago, 1908. 



214 DIAGNOSTIC METHODS. 

The total nitrogen of the urine may be taken as a direct index of the 
protein metabolism. Upon a starvation diet, or one from which the nitroge- 
nous factors have been eliminated, we find a gradual reduction in the amount 
of urinary nitrogen. From about the fourth day of starvation the excretion 
becomes practically constant and continues until severe tissue-decomposition, 
as a sign of impending death, occurs. If, at this time, a diet rich in nitrogen 
be given, a certain amount of the intake will be retained, but not all. If this 
increased nitrogenous diet be continued a certain portion will be retained 
each day until the system again assumes a condition of nitrogenous equilibrium, 
the output equaling the intake. It is to be remembered in this connection 
that the carbohydrates and fats of the diet both have a certain direct influence 
in diminishing the protein metabolism. 

The normal amount of total nitrogen of the urine, with the subject upon 
a mixed diet, varies between 10 and 16 grams per day. The work of Chit- 
tenden 1 has proven that this is much too high for individuals who desire 
to get the most out of their system with the least possible work. In other 
words "physiological economy" is much better subserved by a diet yielding 
from 5 to 6 grams of total nitrogen. The subjects of his experiments showed 
normal activities, both mental and physical, and at the end of the experiments 
felt much better than before them and had gained in weight. Folin 2 in his 
work has shown that the normal excretion of six subjects, each observed for 
a period of five days, was 16 grams of total nitrogen on a diet of 119 grams 
of protein yielding 18.9 grams of nitrogen. On a nitrogen-free diet the excretion 
averaged 3.6 grams. He says: "It may, therefore, be positively stated, as a 
principle in the chemistry of metabolism, that the distribution of the nitrogen in 
urine among urea and the other nitrogenous constituents depends on the absolute 
amount of total nitrogen present." 

The subject of the distribution of the various nitrogenous products of 
the urine has been much changed by this work -of Folin and of Chittenden. 
This distribution or, as it is better called, the " nitrogen partition of the urine," 
varies according to the diet. The following table shows the excretion of the 
various nitrogenous constituents and their percentage relations to the total 
nitrogen under a mixed diet and under one which is nitrogen free. 

Excretion in grams. Percentage of total N. 

Mixed N-Free Mixed N-Free 





Diet. 


Diet. 


Diet. 


Diet. 


Nitrogen, 


16. 00 


3.60 


100. 00 


100. 


Urea N, 


13.90 


2. 20 


86.87 


61. 7 


Ammonia N, 


0. 70 


0. 42 


4-37 


"•3 


Creatinin N, 


0.58 


0. 60 


3- £>3 


17. 2 


Uric Acid N, 


0. 12 


0. 09 


o-75 


2.5 


Undetermined N, 


0. 70 


0. 29 


4-37 


7-3 



1 Physiological Economy in Nutrition, New York, 1905; Mendel, Jour. Am. Med. Assn., 
1914, LXIII, 819; Benedict, Jour. Biol. Chem., 1915, XX, 263; Pepper and Austin, Ibid., 191;, 
XXII, 81; Osborne and Mendel, Ibid., 241. 

2 Loc. cit. 



THE URINE. 215 

It will thus be seen that the total nitrogen excretion of the urine is made 
up of several factors. The principal points to be gained from the above 
table are that the urea, on a nitrogen-free diet, is markedly reduced. This 
reduction must naturally be made up by increase in the other factors. We 
find the ammonia and especially creatinin markedly increased from the per- 
centage standpoint, although both are absolutely diminished. We have, 
therefore, a distinctly endogenous nitrogen metabolism as well as an exogenous 
one. The urea content should, consequently, be considered as of direct 
importance in estimating the degree of protein tissue metabolism, although 
not as usually taught. This urea output cannot at the present time be con- 
sidered as representing from 85 to 90 per cent, of the total endogenous nitrogen, 
but should be regarded more properly as representing between 60 and 65 per 
cent. It is true that the urea as found under mixed diets gives this higher 
figure, but at least 20 per cent, of this must be placed against useless activity 
on the part of the system. In other words, the intake of sufficient nitrogen 
to yield a urea excretion amounting to 85 per cent, of the total N must be 
considered unnecessary. This is a somewhat enlarged expression of the fact 
that most people eat much more than is utilized by the system. 1 

It is seen, therefore, that in the general run of urine examinations the 
urea output does not represent to us the extent of the tissue metabolism, 
as ordinarily we have not sufficiently controlled the diet. The above table 
presents a remarkable percentage increase in the amount of creatinin. The 
figures of Folin show that the absolute quantity of creatinin eliminated, whether 
upon a nitrogen-rich or on a nitrogen-free diet, is remarkably constant for 
the same individual. Although this is influenced to a slight extent by the diet, 
it is so slight that it can be disregarded (see Creatinin). 
Physiologic Variations. 

An increase in the total nitrogen is observed after a heavy protein meal. 
As previously stated, in starvation the nitrogen of the urine becomes constant 
after about the fourth day. This nitrogen excretion observed in starvation is, 
however, less than the minimum amount that must be given in the form of 
protein in order to maintain a nitrogenous equilibrium. As a rule, the amount 
of protein taken in is much in excess of the requirement of the system, so that 
the amount excreted probably represents protein which has never become 
a part of the system. For this reason the fallacy of considering urea as a 
direct representative of the protein metabolism of the tissues becomes evident. 
The intake of protein is, in reality, readjusted to suit the actual needs of the 
body, so that the urea can represent only a portion, about 60 per cent., of the 
total metabolic activity of the tissue protein. A physiological increase in 
the excretion of nitrogen is observed in the infant for four or five days after 
birth. The nitrogen excretion is increased when the intake of water has 
been greater than normal. This fact should be borne in mind in metabolic 
experiments in which the intake of water should be quite as much regulated 
and as well known as the intake of other substances. 

Physiologically, a diminished output of nitrogen is observed on a low 
1 See Mendel and Lewis, Jour. Biol. Chem., 1013, XVI, 19, 37 and 55. 



2l6 DIAGNOSTIC METHODS. 

nitrogen diet and also on a diet rich in carbohydrates and fat, as these latter 
substances provide the greater part of the necessary energy. The system 
must then utilize its own protein. Increased exercise is supposed to give 
a slight increase in the nitrogen output owing to increased muscular activity, 
but' it is to be said that the loss of water through the increased perspiration 
may be such a factor in diminishing the urinary nitrogen that no increase may 
be observed. Certain medicaments as quinin and opium will usually diminish 
the output of nitrogen. 

Pathologic Variations. 
Increased Excretion. 

Perhaps the most marked increase in the urinary nitrogen is observed 
in the acute febrile infections. 1 This increase is not due to the temperature 
per se nor is there any parallelism between the urinary nitrogen and the degree 
of temperature. Whether or not this increase in nitrogen can be directly 
traced to the effects of the toxins produced by the organisms causing the 
disease is still unsettled. That this cannot be the only element is proven 
by the fact that Krehl and Matthes have shown that more protein is destroyed 
in the so-called aseptic fever than is the case in the normal organism under 
similar conditions, dietetic and physical. In some febrile conditions we find 
that the elimination of nitrogen may be reduced during the febrile period, 
while about the time of crisis a very marked output of urinary nitrogen may 
be observed. This is the well-known epicritical elimination of nitrogen. 
In the fever associated with acute nephritis the urinary nitrogen is not increased, 
but is rather diminished owing to the renal insufficiency as well as to the edema 
which occurs. A toxogenic decomposition of protein is found in cases of 
carcinoma, pernicious anemia, chronic tuberculosis, leukemia, scurvy, and 
especially in exophthalmic goiter. In cases of acute yellow atrophy and 
phosphorous poisoning the total nitrogen may be increased, but the percentage 
of urea will be very much diminished. 

In cases of diabetes mellitus the nitrogen excretion is usually much in- 
creased, due more to the effect of the increased nitrogenous diet than to in- 
creased endogenous protein metabolism. Likewise, in diabetes insipidus 
a large increase in urinary nitrogen may be observed. An increase is oc- 
casionally observed in cases of nephritis owing to the large albumin content of 
the urine. During the progress of absorption of an exudate a very high ex- 
cretion may be observed, the resolution of a pneumonic exudate, for instance, 
being easily followed by the variations in the urinary nitrogen. 2 
Diminished Excretion. 

A diminution in the nitrogen excretion is usually observed in convalescence 
from acute and chronic conditions. This is probably due to the attempt 
on the part of the system to make up for the losses incurred during the active 
progress of the disease. It may be diminished in conditions in which the 
absorptive power of the intestine is much reduced. If the oxidative powers 

1 See Sharpe and Simon, Jour. Exper. Med., 1914, XX, 282. 

2 See Matthews and Miller, Jour. Biol. Chem., 19 13, XV, 87; also, Gammeltoft, Habilita- 
tionschr., Kopenhagen, 19 13. 



THE URINE. 217 

of the system are very much reduced as the result of chronic conditions, the 
urinary nitrogen will usually be much diminished. In cases of nephritis, 
both acute and chronic, a large diminution in the urinary nitrogen is observed. 
This is due primarily to the renal insufficiency and to the associated dropsy. In 
such conditions a marked increase may be observed in the fecal nitrogen, 
especially when marked diarrhea is a complicating factor. These periods 
of retention of nitrogen in nephritis may alternate with periods of increased 
elimination, so that examinations at different periods may show greatly con- 
flicting results. When the water output of the urine is largely diminished, 
as a result of transudation, of exudation, or of increased perspiration, the total 
nitrogen may be reduced. 1 

Estimation of Total Nitrogen (Kjeldahl). 

The principle of this method is as follows : 2 The nitrogenous constituents 
of the urine are oxidized by various oxidizing agents into ammonia. This 
ammonia is converted into ammonium sulphate by the sulphuric acid which 
is added at the same time as the oxidizing agents. After the preliminary 
decomposition and oxidation of the organic nitrogen into ammonium sulphate, 
free ammonia may be liberated by the action of strong sodium hydrate and 
distilled into a standard acid solution. Knowing the strength of the acid 
solution, one may then titrate the remaining acid with a standard alkali solution 
and determine how much ammonia has combined with the acid. One c.c. 
of tenth-normal sulphuric acid, used by the ammonia liberated in the dis- 
tillation, represents 0.00 1 401 gram of nitrogen. 
Technic. 

Five c.c. of urine are accurately measured, either with a pipet or buret, 
into a Kjeldahl flask of Jena glass of 800 c.c. capacity. Ten c.c. of concen- 
trated sulphuric acid and approximately 1 gram of copper sulphate are added, 
the flask placed in a hood and heated over a low flame until white fumes of 
sulphuric acid are given off (Gunning's modification). Five grams of 
potassium sulphate are then added and the mixture heated with an increased 
flame to boiling for one-half to three-fourths of an hour. The solution should 
have lost every trace of a yellowish color and should have become by this 
time a clear bluish-green. The worker should be cautioned regarding the 
fumes given off in this process and conduct his work only in a hood with a 
good draft. It is frequently necessary to wash down the carbon from the sides 
of the vessel by shaking the fluid in such a way that the carbonized material 
is carried down to the bottom of the flask. One should be cautious lest he 
lose some of the liquid in this manipulation, which would not only throw 
out his determination but might result in a very severe burn should any of the 
material fall upon him. The mixture is allowed to cool completely before 
the further steps of the determination can be taken. Most workers advise 
at this juncture the transference of the material from the first flask into a 

iMinot, Bull. Johns Hopkins Hosp., 1914, XXV, 332; Hefter and Siebeck, Deutsch. 
Arch. f. klin. Med., 1914, CXIV, 497; Frothingham and Smillie, Arch. Int. Med., 1915, XV, 
204; Murray, Brit. Med. Jour., 1915, I, 151. 

2 See Folin and Farmer (Jour. Biol. Chem., 1912, XI, 493) for a micro-chemical method 
based on this Kjeldahl process. This method is discussed in the section on Blood. 



2l8 



DIAGNOSTIC METHODS. 



second distilling flask. The writer has convinced himself that this procedure 
is not only unnecessary but is even unwise, as the transference may result 
in slight loss of material. He is, therefore, accustomed to use the same flask 
both for the oxidation and distillation. 

After the mixture has cooled the neck of the flask is thoroughly washed 
with a stream of distilled water so that every trace of material may be carried 
from the neck into the body of the flask. Sufficient additional water is added 
to bring the total up to approximately 250 c.c. A little talcum powder, a 
few pieces of pumice stone, or a few pieces of granulated zinc may then be 
added to prevent bumping of the contents when sodium sulphate separates 
out later in the process. Fifty c.c. of 40 per cent, sodium hydrate are then 
added for every 10 c.c. of sulphuric acid used in the original oxidation. Care 




Fig. 72. — KjeldahPs nitrogen apparatus. 

should be taken in adding this strong alkali that none of it touches the upper 
portion of the neck of the flask. The alkalinized mixture is then shaken 
and connected with a Fresenius bulb which is attached to a Liebig condenser 
as shown in the accompanying cut. The outlet tube passes into an Erlen- 
meyer flask which should contain 50 c.c. of tenth-normal sulphuric acid. 
In cases with abnormally high nitrogen values it may be necessary to use a 
larger quantity than 50 c.c. of standard acid, but the writer has found only 
two instances in over 1,500 determinations in which an increased amount 
was necessary. The connection of the distilling flask to the bulb and con- 
denser should be done rapidly to avoid any possible loss of ammonia. 

The distilling flask is heated slowly at first and the heat increased only 
after boiling has become regular. If heated too quickly, spurting of the 
liquid may occur and traces of alkali be carried into the bulb and thence over 



THE URIXE. 219 

into the standard acid. The distillation should continue until about 150 c.c. 
have been distilled over, which will take from 20 to 30 minutes. 

The writer has observed that bumping of the mixture rarely occurs, 
under the conditions outlined above, before the ammonia is completely driven 
over. This can, however, not be taken as an absolute sign that every trace 
of ammonia has been distilled off. In order to see whether such is the case, 
one must test the outlet tube with a piece of moist red litmus-paper which 
will turn blue in the presence of traces of ammonia. If all ammonia has 
not been given off, the distillation must be continued until such is the case. 
If no more ammonia is being evolved, the distilling flask is disconnected from 
the bulb so that no suction may draw the standard acid into the condenser. 
The connecting bulb is removed and the tube of the condenser washed with 
a spray of distilled water so that any material adhering may be washed into 
the standard acid. The outlet tube is disconnected and washed both inter- 
nally and externally into the standard acid solution. 1 

The standard acid solution is then titrated with tenth-normal sodium 
hydrate solution, using cochineal, methyl-orange, alizarin-red, or methyl-red 
as an indicator. The writer prefers the use of the latter. As each c.c. of 
tenth-normal alkali is equivalent to each c.c. of tenth-normal acid, we sub- 
tract, from the original number of c.c. of acid (50), the number of c.c. of stand- 
ard alkali used to neutralize the remaining standard acid. The difference 
gives us the number of c.c. of standard acid neutralized by the ammonia 
given off in the distillation. As each c.c. of tenth-normal acid is equivalent 
to 0.001401 gram of nitrogen, we multiply this factor by the number of c.c. 
of acid neutralized by the ammonia and obtain the amount of total nitrogen 
in 5 c.c. of urine. This result multiplied by 20 yields, of course, the percentage 
of total nitrogen, which may be changed into the actual total amount of nitrogen 
by multiplying it by the number of hundreds of c.c. in the 24-hour specimen. 
It goes, without saying, that the reagents used in this determination must be 
ammonia-free or, at least, that their ammonia content be known. 
(b). Urea (NH 2 ) 2 CO. 

A discussion of the various factors which have to do with urea excretion 
cannot be taken up at this time. The recent work in the laboratory of Hof- 
meister 2 as well as that of Chittenden and of Folin has shown us conclusively 
that it is no longer possible to consider the rate of urea formation as a direct 
measure of protein metabolism. It is without question true, as Leathes 3 
has said, that the nitrogen, or a great part of it, may be removed from the 
protein, converted into urea, and expelled with the urine before the oxidation 
of the rest of the protein molecule has been started upon; and the fact that 
we can trace in the urine excreted in a given time all or the greater part of 
the nitrogen of the protein taken at a meal, tells us nothing whatever about 
the fate of that part of the protein which contains, it may be, as much as 80 
or 90 per cent, of the total energy of the protein. Further, urea is not a measure 



1 See Dakin and Dudley, Jour. Biol. Chem., 1914, XVII, 2 

2 Lang, Beitrage zur chem. Phys. und Path., 1904, V, 340. 

3 Problems in Animal Metabolism, Phila., 1906. 



/ >• 



220 DIAGNOSTIC METHODS. 

of the true protein catabolism, because a great part of it is formed from nitro- 
gen that has never been beyond the liver; and it is not the measure of the 
protein energy because it is largely derived from protein by reactions which 
leave the energy value of the molecules from which it originates but little 
altered. The importance of the denitrifying and desamidization reactions 
of the tissues must be much more considered in the future than they have 
been in the past. 1 

As usually stated in text-books, urea constitutes from 80 to 85 per cent, 
of the total nitrogen output. It has been customary to figure directly the 
amount of tissue protein which must have been decomposed in order to yield 
this amount of urea. These figures can hardly be taken as conclusive of such 
decomposition. Folin has shown that a definite exogenous as well as an 
endogenous protein metabolism occurs. With a patient upon a nitrogen- 
free diet, the urea constitutes only about 60 per cent, of the total nitrogen. 
This would represent the true endogenous urea formation. A diet which 
requires the patient to eliminate much higher percentages of urea is, there- 
fore, causing increased systemic activity, but is not increasing the direct tissue 
decomposition, as this excess never becomes a part of the system. One of 
the most important laws of protein-metabolism is that the amount of nitrogen 
in the body is not increased by, or not in proportion to, an increase in the 
nitrogen intake. 

The amount of urea excreted on an average diet varies from 15 to 40 
grams. 2 Folin finds this excretion to be, on a diet of 119 grams of protein 
yielding 18.9 grams of nitrogen, 298. grams. On a nitrogen-free diet the 
amount of urea is 2.2 grams. This excretion will, of course, vary, depending 
upon the diet. Von Jaksch states that the excretion of urea bears a definite 
relation to the total nitrogen excretion, so that for clinical purposes direct 
urea determinations may well be dispensed with, as the correct urea-content 
of the urine may be found by multiplying the simple nitrogen of the urine 
by the factor 2. This statement should not be regarded seriously by the 
practitioner as the rule, as Folin shows, would require that 93.3 per cent, of 
the total nitrogen in the urine be in the form of urea. 

Pathologic Variations. 

The pathologic increase in the amount of urea is observed under the 
same conditions as those mentioned under an increase of total nitrogen. Thus 
in febrile conditions, in diabetes mellitus and insipidus, 3 after the resorption 
of an exudate, in maliganant conditions, and in exophthalmic goiter, the 
urea may be markedly increased. 

In conditions associated with destruction of hepatic parenchyma, or with 
a diminished rate of blood-flow through the liver, the urea excretion may 
be very considerably diminished. 4 Thus we find in acute yellow atrophy, 

1 See Henriques and Anderson. Ztschr. f. physiol. Chem., 1913, LXXXVIII, 357; Ibid., 
1914, XCII, 21; Davis, Bull. Johns Hopkins Hosp., 1915, XXVI, 154. 

2 See McLean, (Jour. Exper. Med., 1915, XXII, 212 and 366) for a discussion of the law 
governing the rate of urea excretion. 

3 See Bassler, Jour. Am. Med. Assn., 19 14, LXII, 282. 

4 See Fiske and Karsner, Jour. Biol. Chem., 19 13, XVI, 399. 



THE URINE. 221 

carcinoma, cirrhosis, and phosphorus poisoning that the normal urea of the 
urine is replaced by other nitrogenous constituents. The normal function 
of the liver in converting ammonium compounds and amino acids into urea 
is so markedly interfered with that the urea may completely disappear from 
the urine in such cases. It should be mentioned that the liver is not wholly 
responsible for the conversion of the ammonia and amino acids into urea, as the 
kidney unquestionably plays some role in this process. 1 

In acute nephritis there may or may not be a diminution in the eicretion 
of urea, depending upon the extent of the renal insufficiency. In the chronic 
types of nephritis we find that the urea excretion fluctuates to a great extent, 
periods of increase varying with those of decrease. In the early stages, even 
though large amounts of albumin and casts be present, the urea may be normal, 
while in the later stages it is often greatly diminished. 

A diminished excretion of urea is observed in melancholia and in the 
advanced stages of general paresis, while in epilepsy and hysteria an increase 
or a decrease may be observed. In some cases of diabetes mellitus Hirsch- 
feld has shown that the urea output may be diminished as the result of delayed 
absorption from the intestine. In these cases of diabetes the ammonia of 
the urine may be markedly increased owing to its combination with acid 
bodies and consequent withdrawal from hepatic activity. 

While urea is a very important substance both clinically and chemically, 
it is very rarely tested by qualitative methods in medical work. The writer 
feels, therefore, that a description of the properties of this substance would 
best be learned by consulting works on physiologic chemistry. 

Determination of Urea. 

The methods for the determination of urea are numerous. Many of 
them are inaccurate although giving results clinically of importance. In 
the selection of a method for the determination of urea one must be governed 
entirely by the importance of the urea determination in any specific case. 
It should be said in advance that a urea estimation is absolutely useless unless 
taken in conjunction with the total nitrogen. The general practitioner usually 
insists on knowing both the percentage and total amount of urea excreted 
with utter disregard both for the nitrogen of the intake and of the output. 
If the urea is of any value at all it should be determined with these points 
in view. The prevailing idea is that a percentage output of two is approxi- 
mately normal and he therefore bases his conclusion upon an increase or 
decrease with this as a standard. Not infrequently single voidings of urine are 
examined for the urea output. Such examinations are worse than useless 
and may even be harmful. 

The methods for the determination of urea are distinctly separable into 
those useful for purely clinical purposes and those for the more exact metabolic 
work. The general practitioner, believing as he does in the importance of 
urea as an indicator of systemic activity and excretion, must have a rapid 

i See Folin, Jour. Biol. Chem., 1912, XI, 87; Van Slyke, Ibid., 1913, XVI, 213; Marshall 
and Davis, Ibid., 1914, XVIII, 53;Fiske and Sumner, Ibid., 285; Jansen, Ibid., 1915, XXI, 557. 
Palacios, Am. Jour. Med. Sc, 191 5, CXLIX, 267. 



222 



DIAGNOSTIC METHODS. 



and easy method for estimation of urea. It is to be said, however, that the 
more exact methods would better be applied if reliable conclusions are to be 
drawn. 

Knop-Hiifner Method. 
The principle of this method is the decomposition of urea by means 
of sodium hypobromite and the measurement of the nitrogen evolved. Sodium 
hypobromite acts upon urea according to the following equation: 

CO(NH 2 ) 2 + 3 NaOBr=3NaBr + 2N + C0 2 + 2H 2 0. 

The carbon-dioxide evolved in this reaction is absorbed by the excess of alkali 
used, so that all that is necessary is to measure the amount of nitrogen evolved. 
This can be done by direct measurement or by collection in tubes which are 
so calibrated that each c.c. of nitrogen represents a certain percentage of urea. 

Various forms of apparatus, which are 
termed ureometers, have been advised for the 
estimation of urea by this method. The form 
introduced by Knop and Hufner is probably 
the most accurate, but is too complicated for 
general clinical purposes. As this method 
has absolutely no claim to accuracy it is use- 
less in scientific investigations of the nitrogen 
partition of the urine. For clinical purposes, 
however, it serves as a rough approximation 
of the urea output and is, for this reason, 
largely used by the practitioner. 1 This 
method, applied as in the following discus- 
sion, is given merely because of the fact that 
it is almost the only method of estimating 
urea which can be carried out by the general 
practitioner. 

Doremus Ureometer. 

This instrument is seen in the accompanying cut. The graduations 
of the tube are such that the number of mg. of urea in the i c.c. of urine used 
in the test are directly read off instead of the number of c.c. of nitrogen formed 
in the reaction. As o.oi gram of urea in one c.c. represents i gram per ioo 
c.c, this tube will furnish directly the percentage values of urea. 

The tube is filled with a solution of sodium hypobromite made by add- 
ing i c.c. of bromin to 40 c.c. of 20 per cent, cold sodium hydrate solution. 
This hypobromite solution decomposes after standing for a few days so that 
it is never wise to attempt to keep such a solution for any length of time. 
In the writer's laboratory a stock solution of 20 per cent, sodium hydrate 
is prepared and the bromin added to it only as occasion requires for pre- 
paring fresh solutions. In this way one will always have the material at 
hand and need have no fear of his stock solution decomposing. After filling 

1 See Krogh, Ztschr. f. physiol. Chem., 19 13, LXXXIV, 379; Robinson and Mueller, Jour. 
Am. Med. Assn., 19 14, LXII, 514. 




Fig. 73. — Doremus ureometer. 



THE URINE. 



22 3 



r~^\ 



the tube with this hypobromite solution, 1 c.c. of urine is added by means of 
the curved pipet accompanying the instrument. In injecting the urine into 
the solution, the curved end of the pipet should be passed well under the 
curve of the bulb, the tube tilted slightly forward, and the urine forced into 
the hypobromite solution with a slow steady pressure. An evolution of gas 
will be observed at once and will cease in a short time. The carbon dioxid 
given off by the reaction of the urea upon the sodium hypobromite is absorbed 
by the excess of alkali and the nitrogen collects in the upper portion of the 
tube. As soon as the evolution of gas has ceased (5 to 10 minutes), the amount 
of urea is read off directly from the calibrations of the tube as previously described. 

In this determination the urine should, 
theoretically, be free from both albumin 
and sugar. Practically, however, these sub- 
stances are never removed as the method 
yields comparative rather than accurate re- 
sults. Indeed, a very delicate reaction for 
albumin is given by this test. One may 
be practically certain of the presence of 
albumin, if the urine, on being injected 
into the solution, forms rather characteristic 
heavy bubbles and if a dense froth collects 
in the upper portion of the tube. This 
froth settles slowly so that the percentage 
reading requires some time. The forms of 
this apparatus which substitute for the 
glass foot a wooden base are much to be 
preferred as they are not so easily broken. 
The modification of this instrument, as in- 
troduced by Hinds, is seen in the accom- 
panying cut. In this form the urine is 
allowed to run in from the smaller gradu- 
ated tube by opening the stop-cock. This 
modification is an advantage, but does not 
yield any more accurate results than does 
the preceding. 

FolinV Method. 

The principle of this method is as follows: At a temperature of 160 
C. crystallized magnesium chlorid (MgCl 2 6H 2 0) boils in its water of crys- 
tallization. If urea be present it is decomposed by this boiling solution into 
ammonia and carbon dioxid. If the conversion be carried out in acid solu- 
tion, the ammonia formed will combine with the acid and may then be 
liberated by alkalinizing the mixture. The ammonia is distilled into a 
standard acid solution and may then be determined as given under the total 
nitrogen. In this process the preformed ammonia as also the trace present 

1 Ztschr. f. physiol. Chem., 1901, XXXII, 504; Ibid., 1903, XXXVII, 548. 




Fig. 74.- 



Doremus-Hinds ureometer. 
(Hawk.) 



224 



DIAGNOSTIC METHODS. 



os|H 



in the magnesium chlorid will also be determined so that a separate estima- 
tion of these factors must be made and subtracted from the total amount. 

Technic. 
Five c.c. of urine are measured into an Erlenmeyer flask of about 200 c.c. 
capacity, 5 c.c. of concentrated hydrochloric acid, 20 grams of crystallized 
magnesium chlorid, a piece of paraffin about the size of a hazelnut and two 
or three drops of a 1 per cent, aqueous solution of alizarin-red are added. 
An especially constructed safety-tube (see cut) is then inserted and the mix- 
ture boiled until the drops flowing back from the safety-tube produce a very 
perceptible bump or hissing sound on coming in contact with the solution (10 

to 15 minutes). The temperature 1 is then 
somewhat reduced and the heating continued 
for one hour. It is important in this process 
that the reaction must not remain alkaline, 
and, therefore, as soon as the material in the 
flask turns red a very few drops of the acid 
distillate in the safety-tube are shaken back 
into the flask. 2 At the end of the hour the 
contents of the flask are washed into a liter 
Kjeldahl flask with about 700 c.c. of water. 
Twenty c.c. of 10 per cent, sodium hydrate 
are then added and the ammonia distilled, as 
described under total nitrogen, into a stand- 
ard tenth-normal solution of sulphuric acid. 
This distillation should be continued until the 
contents of the liter flask are nearly dry or 
till the distillate shows no trace of ammonia 
with litmus paper. This will require about 
one hour. The distillate is then boiled to 
drive off the carbonic acid, is then cooled, 
and titrated with tenth-normal sodium hydrate 
to determine the amount of acid which com- 
bined with the ammonia formed. .Alizarin- 
red or methyl red are used as indicators. 
One c.c. of tenth-normal sulphuric acid is 
equivalent to 0.001704 gram of ammonia 
(NH 3 ), or 0.00 1 40 1 gram of nitrogen. If the 
nitrogen value be multiplied by 2.143 tne result will be the amount of urea 
in the 5 c.c. of urine taken. From the total c.c. of n/10 sulphuric acid 
neutralized must be subtracted the n/10 sulphuric acid values for the pre- 
formed ammonia as well as for the ammonia which may be present as an 
impurity in the 20 grams of magnesium chlorid used. 

1 Folin advises (Handb. d. Biochem. Arbeitsmeth., 1911, V, 2S6) the use of small glass 
bulbs containing solid HgCH as an indicator of the proper temperature of this reaction. This 
substance melts at 153 C., which is the optimum temperature to insure complete decomposi- 
tion of the urea. 

2 See Folin, Jour. Biol. Chem., 1912, XI, 507. 




Fig. 75 — Folin's urea apparatus. 
{Hawk.) 



THE URINE. 225 

It has been found by Schoorl that when carbohydrates and urea are 
heated together they form very stable condensation products (ureids). For 
this reason this method of Folin does not give accurate results with saccharin 
urine. A combination of this method with that of Morner, which will be 
described later, will give absolutely accurate results. In the determination 
of the ammonia values of the preformed ammonia and of the magnesium 
chlorid, the later methods of Folin must be used. 

Morner-Sjoqvist Method. 
This method 1 is an extremely accurate one, but no more so than that of 
Folin, except in saccharin urines. If albumin be present it must, however, 
be removed by heat and acetic acid and the original volume of the urine re- 
stored. If the urine contains large amounts of hippuric acid this method 
may not give accurate results, as Salaskin and Zaleski have shown. 

Technic. 

Five c.c. of urine are placed in a flask with 5 c.c. of baryta mixture con- 
sisting of a saturated barium chlorid solution containing 5 per cent, of barium 
hydrate. One hundred c.c. of a mixture of two parts of 97 per cent, alcohol 
and one part of anhydrous ether are then added and the mixture allowed to 
stand in a closed flask overnight. It is then filtered and the residue washed 
with fresh alcohol and ether mixture and the combined nitrates evaporated 
at a low temperature (6o° C). Urea will be practically the only nitrogenous 
body left in solution, with the exception of traces of ammonia. When the 
evaporated nitrate has been reduced to about 25 c.c. in volume, a few c.c. 
of water and a small amount of calcined magnesium oxid are then added, 
the mixture stirred, and heated to drive off the ammonia; or the residue may 
be treated by Folin's method as previously described. This heating is con- 
tinued until the vapor shows no alkalinity when tested with moistened litmus 
paper, a result usually obtained when about 10 to 15 c.c. of the mixture remain. 
The fluid and the residue are then washed into a Kjeldahl flask and treated 
with concentrated sulphuric acid, copper sulphate, and potassium sulphate 
as in the determination of total nitrogen. One part of nitrogen is equivalent 
to 2.143 grams of urea. 

If much hippuric acid be present, Braunstein 2 advises oxidation of the 
urea by heating the evaporated residue with 10 grams of solid phosphoric 
acid (in an air-bath) to 140 to 145 C. for five hours. Alkalinize and distill 
as previously described. Benedict's 2 newer method also yields accurate results. 2 

Urease Method. 

Marshall has introduced a method for the determination of urea in various 
body fluids, which is based on an entirely different principle from the others 
advocated and which is accurate and extremely simple. 4 He takes advantage 

1 Skand. Arch. f. Physiol., 1891, II, 438; Ibid., 1903, XIV, 247. 

2 Ztschr. f . physiol. Chem., 1901, XXXI, 381. 

3 Jour. Biol. Chem., 1910, VIII, 405. See, also, Milroy, Biochem. Jour., 1913, VII, 399. 
4 Takeuchi, Jour. College Agriculture, Tokio, 1909, I 1; Chem. Ztg., 1911, XXXV, 408; 

Marshal], Jour. Biol. Chem., 1913, XTV, 283 ; Ibid., 1913, XV, 4955 Hahn and Saphra, Deutsch. 
med. Wchnschr., 1914, XL, 430; Van Slyke and Cullen, Jour. Am. Med. Assn., 1914, LXII, 

*5 



226 DIAGNOSTIC METHODS. 

of a fact, first discovered by Takeuchi, that an aqueous extract of soy bean 
(glycine hispida) contains a ferment which converts urea into ammonium carbon- 
ate and which neither acts upon anything else nor is interfered with by anything 
ordinarily found in the various body fluids. In his method Marshall employs 
this aqueous extract, but this has the disadvantage of requiring many hours to 
react quantitatively and, further, of losing its activity on standing for a few days, 
the loss being more rapid the higher the temperature at which it is kept. For 
these reasons the writer prefers to adopt the modification of Van Slyke and 
Cullen. These workers have produced a permanent preparation of this ferment 
by the following method : Extract soy bean meal with 5 parts of water and pour 
this extract into 10 volumes of acetone. Filter off the precipitate and dry it. 1 

This urease dissolves readily in water, forming an opalescent solution. 
"Under given conditions, a given amount of it will decompose a definite amount 
of urea per minute and no more, regardless of how much excess urea may be 
present. The preparation is most active at a temperature approximately 55 C. 
(13 1° F.) and in a perfectly neutral solution. Between 10 and 50 C. (50 and 
122 F.), increasing the temperature over any io° interval doubles the rate at 
which the preparation acts. Heavy metals, or acid in a relatively slight con- 
centration, destroy the activity. The urease is not quite so sensitive toward an 
alkaline reaction, but its activity is decreased by even weak bases. Ammonium 
carbonate depresses the activity to about one-eighth the optimum, so that the 
action of the enzyme is self-retarding. The retarding effect of the ammonium 
carbonate formed can be neutralized and conditions for optimum activity ob- 
tained by the addition of potassium dihydrogen phosphate and dipotassium 
hydrogen phosphate in equimolecular proportions. This phosphate mixture 
also acts as a stabilizer to solutions of enzyme." 
Preparation of Enzyme Solution. 

Two grams of the enzyme preparation, 0.6 gram of dipotassium hydrogen 
phosphate and 0.4 gram of potassium dihydrogen phosphate are stirred up with 
a rod in 10 cc. of water. The preparation dissolves quickly, forming an opal- 
escent solution. If this solution be covered with toluol it will hold its activity 
for a week or so, but it is better technic to employ relatively fresh solutions. 
Technic 

This method employs a portion of the technic originally introduced for 
determination of ammonia by Folin (see page 230). Dilute 5 cc. of urine with 
water to 50 cc. Mix this thoroughly and transfer, by means of a pipet, 5 cc 
of this diluted urine to a clean, fairly heavy test-tube of about 50 cc capacity. 
Add 1 cc. of the enzyme solution and 1 drop of caprylic alcohol or a few drops of 
benzol to prevent foaming during the later aeration. Close this tube (A) with 
a stopper and allow it to stand 15 minutes in order to permit the enzyme to act 
upon the urea present. 

1558; Jour. Biol. Chem., 1914, XIX, 141 and 211; Van Slyke, Zacharias and Cullen, Deutsch. 
med. Wchnschr., 1914, XL, 1219; Plimmer and Skelton, Biochem. Jour., 1914, VIII, 70; 
Hahn, Deutsch. med. Wchnschr., 1915, XLI, 134; Eigenberger, Ztschr. f. physiol. Chem., 1915, 
XCIII, 370; Jacoby and Sugga, Biochem. Ztschr., 1915, LXIX, 116. 

1 If one does not wish to prepare this urease for himself, he may obtain it from the Arlington 
Chemical Company of Yonkers, N. Y. 



THE URINE. 227 

Into a second test-tube (B) measure with a buret 25 c.c. of fiftieth normal 
(N/50) hydrochloric or sulphuric acid and add 1 drop of a 1 per cent, solution of 
alizarin as an indicator and 1 drop of caprylic alcohol or benzol. 

By means of glass and rubber tubing connect these two tubes in such a way 
that air may be forced through tube A into tube B (see page 230). After tube A 
has stood for 15 minutes, pass the air current for about 1/2 minute in order to 
carry over into the acid any ammonia which may have passed into the upper 
portion of tube A. Now shut off the air, open tube A and pour into it 4 or 5 
grams of dry potassium carbonate. Close tube A at once and pass the air current 
till all the ammonia is driven into tube B. This time depends on the rate at 
which the air is passed. With the ordinary apparatus this varies from 15 to 30 
minutes. 

When this process is finished, titrate the excess of acid in tube B with fiftieth 
normal (N/50) sodium hydrate, titrating to the appearance of the first permanent 
red point. Subtract the number of c.c. of standard alkali used from 25 (the 
number of c.c. of standard acid originally measured into tube B) and multiply 
this result by 0.056 to obtain the grams of urea nitrogen plus ammonia nitrogen 
in 100 c.c. of urine. 

The ammonia nitrogen, which is not referable to the decomposition of urea, 
is obtained by the same method as the above, using 5 c.c. of undiluted urine 
and adding no urease solution. In this latter case multiply the number of c.c. 
of excess acid by 0.0056 instead of 0.056 to determine the percentage of ammonia 
nitrogen originally present in the urine. Subtract this latter figure from the 
figure for the combined nitrogen of urea and ammonia and obtain the true 
figure for the ammonia nitrogen derived from the action of the urease. If this 
nitrogen figure be multiplied by 2.143, one obtains the percentage of urea present 
as such. 1 

(c). Ammonia (NH 3 ). 

This substance, although chemically belonging in the class of inorganic 
compounds, is so closely related to the nitrogen metabolism that it is more 
properly discussed under the heading of Nitrogenous Bodies. 

Ammonia is one of the most important products of protein metabolism. 
It is constantly present in small amounts in normal urine averaging about 
0.85 gram of NH 3 in 24 hours, representing from 4 to 5 per cent, of the total 
nitrogen. It is present in combination with various acids and may represent 
largely a portion of the nitrogen which has not been transformed into urea, 
but has been used to combine with acid substances formed in the protein 
metabolism of the body. Any increase in the production of acid in the system 
or any increased intake of noncarbonate-forming acids will lead to an increased 
excretion of ammonium salts. This is an important factor in the metabolism 
of conditions associated with an acidosis. 

The total output of ammonia will vary under normal conditions with 

1 Instead of using the method of aeration, one may titrate directly the ammoniurn carbonate 
formed by the action of the urease on the urea, using somewhat larger amounts of urine (5 c.c.) 
and titrating with N/10 hydrochloric acid, with methyl-orange as an indicator. This was the 
original method of Marshall, but it does not yield as accurate results as the technic described. 



228 DIAGNOSTIC METHODS. 

the diet or, in other words, with the intake of total nitrogen. 1 While the 
increase of the total nitrogen of the urine on increased nitrogen intake is largely 
in the form of urea, yet a small increase in the absolute amount of ammonia 
must occur. Likewise we observe a diminished intake of nitrogen reducing 
the absolute value of ammonia, but largely increasing its relative value. Thus 
Folin finds with a total excretion of 16 grams of nitrogen, an ammonia output 
of 0.85 gram (4.3 per cent.); while on a nitrogen-free diet a total nitrogen 
output of 3.6 grams was observed with an ammonia elimination of 0.51 gram 
(11.3 per cent.). We therefore conclude with Folin as follows: "With pro- 
nounced diminution in the protein metabolism (as shown by the total nitrogen 
in the urine), there is usually, but not always, and therefore not necessarily, 
a decrease in the absolute quantity of ammonia eliminated. A pronounced 
reduction of the total nitrogen is, however, always accompanied by a relative 
increase in the ammonia nitrogen, provided that the food is not such as to 
yield an alkaline ash." 

Although the ammonium salts of many organic acids are converted into 
urea in the system we find the ammonium salts of the sulphuric and phosphoric 
acids formed in the decomposition of protein material are excreted as such. 
Moreover, we observe that an increased consumption of fat, either taken 
in as food or derived from the tissue, is associated with a combination of am- 
monia with the fatty acids. This provision of metabolism, by which the 
system is protected against the deleterious effects of increased acidity by 
neutralization of acid compounds with ammonia, is of the greatest importance, 
as the fixed alkalies of the tissues are thereby maintained in their usual con- 
centration unless the pathologic processes be extreme. 

Pathologic Variations. 

An increased output of ammonia is observed in cases of diminished oxida- 
tive powers of the system, in febrile diseases, in hepatic disturbances such 
as carcinoma and acute yellow atrophy, in. uremia, in acid intoxication, in 
dyspnea from any cause, in the toxic vomiting of pregnancy, in delayed chloro- 
form poisoning, and especially in diabetes mellitus. In this latter condition the 
degree of acidosis may be conveniently followed by watching the ammonia 
output. 

A reduction in the amount of ammonia is observed in many cases of 
nephritis and in some cases of carcinoma of the stomach, although there 
is at the same time a diminished excretion of hydrochloric acid in the gastric 
contents. Edsall reports a reduction in cases of periodic insanity preceding 
the attack, while a rise is observed as the attack proceeds. Administration 
of large doses of the fixed alkalies will usually diminish the ammonia. output. 2 

Quantitative Determination of Ammonia. 
Like the methods given under Urea, many have been advanced for the 
determination of the urinary ammonia. One of these, though inaccurate, 
has been so long used and even to-day is so relied upon in many quarters that 

1 See Tanji, Deutsch. Arch. f. klin. Med., 1914. CXYI, 92. 

2 See Janney, Ztschr. f. physiol. Chem., 1912, LXXYI, 99. 



THE URINE. 



229 



the writer includes it with the understanding that he advocates only the ac- 
curate methods. If the ammonia is worth determining, definite results should 
be sought and hence the most accurate methods are the ones to be used. They 
are no more complicated, not as time-consuming, and give more reliable 
results. 

Method of Schlbsing. 
This method is the one most commonly used, but is open to the objection 
that it does not yield accurate results and is time-consuming, but has the 
advantage of simplicity. It is argued by many that the element of time is of 
no importance, as clinically one would not wait for an ammonia determination 
before instituting vigorous treatment. On the other hand, in metabolic work 
it is of a great advantage to get the work out of the way as quickly as is possible 
and consistent with accurate results. 

Technic. 

Twenty-five c.c. of urine are placed in the vessel B (preferably a Petri dish) 
(see cut). Above this is placed a glass triangle upon which rests a dish (C) 




Fig. 76. — Schlosing's ammonia apparatus. 

containing 20 c.c. of tenth-normal sulphuric acid. Twenty c.c. of milk of 
lime are then poured into the dish containing the urine and the whole covered 
with a bell-jar, the borders of which have been well greased to make an air- 
tight union when the jar is placed upon the glass plate. This apparatus 
is then allowed to stand at room temperature from four to five days, during 
which time the ammonia, liberated by the action of the milk of lime upon 
the ammonium salts of the urine, will be taken up by the sulphuric acid in 
the vessel C. At the end of this time the bell- jar is removed, the acid titrated 
with tenth-normal sodium hydrate, and the number of c.c. of remaining acid 
determined. One c.c. of tenth-normal sulphuric acid neutralized by the 
evolved ammonia represents 0.001704 gram of ammonia. This figure is 



23° 



DIAGNOSTIC METHODS. 



multiplied by 4 to obtain the percentage ammonia value. If any moisture is 
present on the inside of the bell-jar it should be washed into the sulphuric 
acid before titration. 

This method, as previously stated, does not give accurate results owing 
to the fact that ammonia may be split off from urea and thus give figures 
which are somewhat high. It has been found that if the apparatus be kept 
at 38 C, the time necessary for this reaction may be reduced to 48 hours. 
If we add to the urine instead of the milk of lime, 0.5 gram of sodium carbonate 
and about 10 grams of sodium chlorid, no ammonia will be split off from the 
urea and no decomposition of the urine will occur (Schaffer). 




Fig. 77. — Folin's ammonia apparatus. (Haivk.) 

Folin's Method. 

The ammonia in this method 1 is set free by the addition of a weak alkali 
(sodium carbonate), is then removed from the urine at ordinary room tem- 
perature by means of a strong air-current, is collected in tenth-normal sulphuric 
acid and then titrated. 
Technic. 

Twenty-five c.c. of urine are poured into an aerometer cylinder (30 to 40 
cm. high), 15 grams of potassium oxalate, 2 1 gram of dry Xa 2 C0 3 and some 
crude petroleum (to prevent foaming) are added. The upper end of the 
cylinder is then closed by means of a doubly perforated rubber stopper, through 
which pass two glass tubes, only one of which is long enough to reach below the 
surface of the liquid. 3 The shorter tube (about 10 cm.) in length is connected 

1 Ztschr. f. physiol. Chem., 1902, XXXVII, 161; Ibid., 1903, XXXIX, 477. 

2 Jour. Biol. Chem., 19 10, VIII, 497. 

3 Folin and Macallum (Jour. Biol. Chem., 1912, XI, 523) have originated a micro-chemical 
method, which permits of much quicker estimation. 1 to 5 c.c. of urine, depending on the 
amount suspected, are placed in a large Jena test-tube by means of an Ostwald pipet. Add 
to the urine a few drops of a solution containing 10 per cent, potassium carbonate and 15 per 
cent, potassium oxalate and follow this with a few drops of kerosene or benzol to prevent 
foaming. Pass the strong air current for 10 minutes, collecting the ammonia in a 100 c.c. 
volumetric flask containing about 20 c.c. of water and 2 c.c. of X/10 hydrochloric acid. Nes- 
s]erize this solution against a standard ammonium sulphate solution as described in section 
on Blood or titrate it with N/50 alkali using methy]-red as an ndicator. 



THE URINE. 



23I 



V- 



cife 



with a calcium chlorid tube filled with cotton, which in turn is connected 
with a glass tube extending to the bottom of a wide-mouthed bottle (capacity 
about 500 c.c.) which contains 20 c.c. of tenth-normal sulphuric acid, 200 c.c. 
of water and a few drops of an indicator (alizarin-red). The complete ab- 
sorption of the ammonia by the sulphuric acid is most easily insured by the 
use of a simple absorption tube which compels a very intimate contact of the 
air coming from the cylinder with the acid and water in the absorption bottle. 
This absorption bulb consists of a glass tube, measuring about 8 mm., in 
diameter, one extremity of which has been blown into a small bulb. By 
means of a heated platinum wire, 10 or 12 holes, each about 1 mm. in diameter, 
are made in this bulb. 

The absorption bottle is then attached by means of a glass tube and 
rubber connection to a filter-pump which will draw 700 liters of air per hour. 1 
The air passing through the alkaline urine will draw the ammonia into the 
standard acid in from one and one-half to two hours. In order to exclude 
any error due to the presence of ammonia in the aspirated air, 
a similar absorption apparatus is attached to the distal side of 
the evolution flask. The amount is then determined by titra- 
tion of the standard acid with tenth-normal sodium hydrate, 
using methyl-red as an indicator. Steel, Gies and others advise 
the use of XaOH instead of Xa 2 C0 3 in this method on the 
ground that the latter will not liberate XH 3 from ammonium 
magnesium phosphate. This would not seem to militate against 
the method as the triple phosphate is rarely, if ever, present 
in the urine in appreciable amounts except when the urine is 
decomposed. Under such circumstances the ammonia factor 
would be useless from the metabolic standpoint. 
Formalin Method. 

This method, originated by Ronchese 2 and Malfatti, 3 de- 
pends on the fact that a solution of an ammonium salt, treated 
with formaldehyd, decomposes with the formation of hexa- 
methylen-tetramin, the acid combined with the ammonia being 
liberated. This can then be determined by titration. 

Dilute 10 c.c. of urine with 50 c.c. of water, add 2 or 3 
drops of a 1 per cent, alcoholic solution of phenolphthalein and 
neutralize with N/10 XaOH. Five c.c. of formalin, previously 
neutralized with N/10 XaOH, are added to the neutralized urine and the mix- 
ture is again titrated with X/10 XaOH to the appearance of a faint, permanent 
pink color, the amount of alkali used being noted. It is evident that 1 c.c. of 
this X/10 XaOH is equivalent to 1 c.c. of X/10 XH 3 or, in other words, repre- 

1 In laboratories in which compressed air is available, the ammonia may be forced from the 
generating cylinder into the cylinder containing the standard acid. In this case, a cylinder 
containing 100 c.c. of 10 per 'cent, sulphuric acid is interposed between the air blast and the 
generating cylinder in order to catch any ammonia which may be present in the air. 

2 Jour. Pharm. et Chim., 1907, XXV, 611; Bull. Soc. Chim. de France, 1907, I, 900. 
3 Ztschr. f. anal. Chem., 1908, XLVII, 273. 



Fig. 78 — 
Folin's absorp- 
t io n bulb. 
(Hawk.) 



232 



DIAGNOSTIC METHODS. 



sents 0.001704 gram of NH 3 . Multiply the number of c.c. of N/10 NaOH 
used by this factor and obtain the NH 3 in 10 c.c. of urine. 

(d). Uric Acid, C s H 4 N 4 3 . 

In times past uric acid has been credited with much more clinical im- 
portance than is to-day ascribed to it. It is not, as many believe, a product of 
protein decomposition, as such, but can be derived only by the splitting of the 
nucleo-proteins. These nuclein bodies are compounds of protein with nucleic 
acid, the latter constituent splitting up into thymic acid and derivatives of 
"purin," among which we find uric acid, xanthin, hypoxanthin, etc. 

Uric acid, as well as the purin bases to be discussed later, are derivatives 
of Fischer's hypothetical purin nucleus, with the formula C 5 H 4 N 4 and the 
following graphic structure. The figures in brackets represent the number 
of the atom or group of atoms : 



(1) 



N 



CH 



(2) HC 



(5) 
C- 



(6) 

(7) 
-NH 



(4) 
-C- 



CH (8) 
-N 



(3) N— 

Each of the purin derivatives is formed by the replacement of one or 
more of the hydrogen atoms in this nucleus by various atoms or groups of 
atoms. Thus we find that uric acid is a derivative in which 3 atoms of 
oxygen have been substituted for the hydrogens in positions 2, 6, and 8. The 
hydrogen atoms are not replaced, but shift in the direction of the double 
bonds, passing to the nitrogen atoms 1, 3, and 9. The structure of uric acid 
with the formula C 5 H 4 N 4 3 is 



HN- 



-CO 



OC 



HN- 



C- 



-NH 



CO 



-NH 



Its chemical name is, therefore, 2, 6, 8, tri-oxy purin. 

Uric acid is derived from the nucleins of the food, as well as from the 
nucleins of the tissues. 1 We can conceive, consequently, that we may have a 
uric acid excretion which may be, in part, referable to each of these factors. 
This is the basis of the theory of endogenous and of exogenous purin metabolism, 
the former representing the uric acid formation from the tissue nucleins, the 
latter that from the food directly. It is self-evident that the exogenous purin 

1 See Thannhauser, Ztschr. f. physiol. Chem., 1914, XCI, 329; Thannhauser and Boomes, 
Ibid., 336; Bass, Arch. f. exper. Path. u. Pharmakol., 1914, LXXVI, 40; Taylor and Rose, 
Jour. Biol. Chem., 1914, XVIII, 519. 



THE URINE. 233 

metabolism, being directly dependent upon the diet, will vary more than will 
the endogenous form. This latter type is dependent not only upon the 
metabolism of the various cellular elements of the body, but also upon the 
degree of direct synthesis of uric acid in the system, as well as upon the extent 
of conversion of uric acid into urea. This latter statement must not be inter- 
preted as meaning that uric acid is simply a stage in the conversion of all 
protein into urea. This idea, which held sway for so long, has fortunately 
been abandoned. However, we do know, from the work of Frerichs and Woh- 
ler that the system does transform a certain amount of uric acid into urea. 

Burian and Schur point out that the uric acid eliminated by man on 
a purin-free diet (endogenous uric acid metabolism) is for each individual 
a constant quantity and entirely independent of the total amount of nitrogen 
eliminated. This fact has so firmly fixed itself in the minds of the profes- 
sion that the important results of Folin have been largely unnoticed. This 
latter worker finds that "when the total amount of protein metabolism is 
greatly reduced, the absolute quantity of uric acid is diminished, but not 
nearly in proportion to the diminution in the total nitrogen, and the per cent, of 
the uric acid nitrogen in terms of the total nitrogen is, therefore, much in- 
creased." It would seem, therefore, that this point is still very debatable. 
Quoting again from Folin "if the endogenous uric acid is to be considered as 
derived from the cell nucleins exclusively, it would, indeed, seem highly plaus- 
ible that the quantity should tend to remain constant, even with very great 
variations in diet. Rigid proof that the endogenous uric acid elimination 
is for each individual a constant quantity would be strong evidence in favor 
of such a theory. Burian and Schur support the view that the endogenous 
uric acid is derived from the cell nucleins, but they contend that in man about 
one-half of the uric acid so derived is destroyed inside the organism and that 
only the other half is eliminated. With the introduction of this important 
modification of the nuclein theory, there is no longer any reason why the uric 
acid elimination should not be a decidedly variable factor which might well 
be susceptible to change under the influence of many different changes in the 
conditions, among others, changes in diet." It will be seen, therefore, that one 
must be on his guard in drawing conclusions from the amount of uric acid in 
the urine, as to the degree of nuclein decomposition within the system. 1 

Physiologic Variations. 

The output of uric acid varies, depending upon the diet, from 0.2 to 2 
grams in 24 hours. On a diet of 119 grams of protein with a total urinary 
nitrogen output of 16 grams the uric acid eliminated was 0.37 gram (0.8 per 
cent, of total N); while on a nitrogen-free diet with a urinary nitrogen of 3.6 
grams the output of uric acid was 0.09 gram (2.5 per cent, of total N). It is 
increased physiologically by increase in the nucleins of the diet, sweet-breads, 
liver, kidneys, and brain yielding very large amounts of uric acid. A meat 
diet will lead to a larger excretion of uric acid than will a vegetable diet, the 

1 See Siven, Arch. f. d. ges. Physiol., 1912, XCLV, 283; Ibid., 1914, CLVII, 5S2; Faustka, 
Ibid., 1914, CLV, 523; Raiziss, Dubin and Ringer, Jour. Biol. Chem., 1914, XIX, 473; Daniels 
and McCrudden, Arch. Int. Med., 1915, XV, 1046. 



234 DIAGNOSTIC METHODS. 

maximum output being observed about five hours after a hearty protein meal. 
According to Horbaczewski, this increase is dependent upon the leucolysis 
which occurs at the time of the disappearance of the digestive leucocytosis. 
The amount of exercise will also influence the output in the urine; while an 
intake of a large amount of water will increase the normal uric acid value. A 
certain relation between the amount of urea and of uric acid excreted seems 
to obtain. As a rule, it may be said that the nitrogen of the urea is to the nitro- 
gen of the uric acid as 50 or 60 to 1. It is probable that variations in this rela- 
tion have at present much less clinical value than in times past. It does not 
seem to the writer consistent to assume a special "uric acid diathesis" if this 
normal relation be disturbed in the sense that the uric acid is increased. 

Pathologic Variations. 

A pathologic increase of uric acid is observed whenever we have increased 
protein catabolism. Thus, in fever, the output of uric acid runs parallel to that 
of urea. In cases associated with marked leucocytosis a large increase in the 
uric acid output may be noted, which is referable to the constant leucolysis. 
This increase is especially marked in leukemia, an output of more than 12 grams 
in 24 hours having been observed by Magnus Levy. In cases of pneumonia, 
associated with a high leucocyte count, an increase in the uric acid output is 
seen, being especially marked after the crisis, but may even precede the crisis. 

Gout has so long been associated, in the minds of the profession, with 
increased uric acid formation that such a relationship is generally accepted. 
It is true that during the acute attack the blood may contain an increased 
amount of uric acid, but never in such a large amount as in cases of leukemia, 
for instance. The mere excess of uric acid in the blood can, therefore, not be 
the determining factor. As our knowledge of the true etiology of gout is so ob- 
scure the writer will not attempt a discussion of uric acid in such relations. 
According to Futcher the uric acid is below the normal standard preceding 
the acute attack, rises to much increased values during the attack, and again 
falls below the normal limit after the subsidence of the acute symptoms. In 
acute articular rheumatism an increased elimination is noted during the febrile 
period, a decrease being observed as convalescence approaches. In diabetes 
mellitus an increase or a decrease may be observed, the uric acid excretion 
varying inversely as that of the sugar, giving rise to the term diabetes alternans. 
In cases showing much degeneration of hepatic tissue, as in cirrhosis and acute 
yellow atrophy, uric acid may be largely increased. 

A diminished excretion of uric acid is usually observed in the ordinary 
forms of anemia while in pernicious anemia an increase may be noted. Chronic 
interstitial nephritis, chronic lead-poisoning, purpura hemorrhagica, and some 
cases of epilepsy, are associated with a diminished output of uric acid. Large 
doses of quinin or of opium may diminish the uric acid, while salicylic acid, 
chinic acid, colchicin, urotropin, piperazin, nucleinic acid, etc., may increase 
the amount. 

The chemical properties of uric acid may be found in works on physio- 
logic chemistry. Qualitative tests are frequently desirable for the detection of 



THE URINE. 235 

the nature of certain deposits, as, for instance, renal infarcts. These will be 
discussed in a later section in association with the description of the various 
crystalline forms which uric acid may assume in a urinary deposit. 

Quantitative Determination. 
Folin's Method. 
This modification 1 of 'Hopkins' method depends upon the precipitation of 
uric acid, as ammonium urate, by addition of ammonium sulphate. The urate 
is decomposed with sulphuric acid and the liberated uric acid is determined by 
titration with a standard solution of potassium permanganate. 

Technic. 

Two hundred c.c. of urine are treated with 50 c.c. of a reagent consisting of 
500 grams of ammonium sulphate, 5 grams of uranium acetate, and 60 c.c. 
of 10 per cent, acetic acid, dissolved in 650 c.c. of water. The mixture is al- 
lowed to stand without stirring for about one-half hour. The precipitate of 
uranium phosphate has then settled and the clear supernatant liquid is re- 
moved by siphonage or by decantation. One hundred and twenty-five c.c. of 
this clear fluid, representing 100 c.c. of urine, are measured into a beaker, 5 c.c. 
of strong ammonia are added, and the mixture set aside until the following day. 
The precipitate of ammonium urate, which is produced by alkalinizing the solu- 
tion saturated with ammonium sulphate, is then filtered off, and washed with 10 per 
cent, ammonium sulphate solution until the filtrate is practically free from chlorids. 
The filter is removed from the funnel, opened, and the precipitate rinsed back 
into the beaker, enough water to make about 100 c.c. being used. The pre- 
cipitate is now dissolved by adding 15 c.c. of concentrated sulphuric acid. The 
solution is then titrated with a twentieth-normal solution of potassium perman- 
ganate, until the first pink coloration is observed extending through the entire 
liquid after the addition of two drops of the permanganate solution. Each c.c. 
of potassium permanganate used corresponds to 3.75 mg. of uric acid. Owing 
to the solubility of the ammonium urate, a correction of 3 mg. per 100 c.c. of 
urine must be made. The corrected result gives the percentage of uric acid. 2 

A method of making a twentieth-normal potassium permanganate solution 
may be found in any work in general chemistry. The student is to be cautioned 
that a twentieth-normal solution of potassium permanganate, as used in 
this connection, has reference to one of such a concentration that one liter 
would contain 0.05 gram of available oxgyen for oxidizing purposes. This 
solution is obtained by dissolving 1.581 grams of pure KMn0 4 in one liter of 
water. As this weighing may not be sufficient, owing to slight impurities in the 
permanganate, it is wise to titrate this solution against a known twentieth- 
normal solution of oxalic acid. If the solutions correspond, 1 c.c. of each 
should be the equivalents. The titration of the oxalic solution is made by meas- 
uring out 10 c.c. of oxalic acid solution, diluting to approximately 100 c.c. with 
distilled water, and adding 15 c.c. of concentrated sulphuric acid. The per- 

1 Ztschr. f. physiol. Chem., 1901, XXXII, 552. 

2 Skorczewski und Sohn (Wien. klin. Wchnschr., 1911, XXIV, 1700) show that the urine 
of patients taking atophan may reduce the permanganate and, hence, introduce an error. 



236 DIAGNOSTIC METHODS. 

manganate solution is then added drop by drop until a faint permanent red color 
is observed throughout the liquid, which does not disappear on stirring but 
persists for at least 30 seconds. If these solutions do not correspond the per- 
manganate solution must be diluted according to the formula previously given 
under the determination of the chlorids in the urine. 

Salkowski-Ludwig Method. 

This method is not as convenient as that of Folin, but is inserted as it serves as 
a combined method of determining uric acid and the purin bases at the same time. 

The principle is as follows: Uric acid is precipitated, in the form of a 
double urate of silver and magnesium, by an ammoniacal silver solution in the 
presence of magnesium salts. The silver is removed by hydrogen sulphid 
and the uric acid precipitated by hydrochloric acid, after which it may be es- 
timated by direct weighing or by determination of the nitrogen. 

Technic. 

Two hundred and forty c.c. of urine are treated with 60 c.c. of magnesium 
mixture, which is made up as follows: One hundred grams of crystallized 
magnesium chlorid and 200 grams of ammonium chlorid are dissolved in 
about 500 c.c. of water, ammonia is added until the mixture smells strongly of 
this substance and the whole is then made up to one liter. The above mixture 
is well stirred and immediately filtered through a dry niter-paper into a beaker. 
Two hundred and fifty c.c. of nitrate (representing 200 c.c. of urine) are then 
measured off and treated with 10 to 15 c.c. of an ammoniacal silver nitrate 
solution (one liter of which contains 26 grams of AgX0 3 and enough ammonia 
to dissolve the precipitate formed). The precipitate should be of a flocculent 
gelatinous nature and of a yellowish color. If the precipitate be white, too 
much silver chlorid is present and more ammonia must be added. The clear 
solution above the precipitate should contain an excess of silver chlorid which 
may be shown by adding a little nitric acid to a few drops of the clear super- 
natant liquid. This mixture is then filtered, any particles adhering to the 
beaker being transferred to the filter by means of water and a rubber-tipped 
glass rod. The residue on the filter is washed with distilled water until the wash 
fluid shows no trace of silver or of chlorids. The funnel is now placed in the neck 
of a liter flask, the tip of the filter-paper is perforated with a glass rod, and 
the precipitate washed into the flask and thoroughly mixed with the water. 
This solution is made faintly acid by the addition of two or three drops of 
hydrochloric acid. Three to four c.c. of 10 per cent, copper sulphate solution 
are then added and the mixture boiled, after which hydrogen sulphid is passed 
through the hot mixture to precipitate the silver salts. This gas should be 
passed until the solution is saturated with it, after which the solution is boiled 
and filtered. The precipitate is washed with hot water and the filtrate, which 
must be clear and colorless, is evaporated to a small bulk (10 to 15 c.c). Ten 
to fifteen drops of hydrochloric acid are then added, the mixture stirred and 
allowed to stand for a few hours, preferably over night. This addition of 
hydrochloric acid precipitates the uric acid and leaves the purin bases in so- 
lution. The crystals of uric acid are filtered off on a small weighed filter, are 






THE URINE. 237 

washed with water slightly acidified with HC1 to such an extent that the total 
wash-water and filtrate should not be more than 50 to 60 c.c. The precipitate 
is washed with alcohol, carbon disulphid, and ether, and is then dried and 
weighed. The difference between the original weight of the filter-paper and 
that of the paper and precipitate is the amount of uric acid in the 200 c.c. of 
urine. Owing to the slight solubility of uric acid in acidulated water, a correction 
of 0.00048 gram must be added for every 10 c.c. of the filtrate and wash-water. 

Instead of weighing the uric acid, the filter-paper and contents may be placed 
in a Kjeldahl flask and a nitrogen determination made as previously described. 
The nitrogen value multiplied by 3 will give the weight of uric acid in 200 c.c. of 
urine. 

The nitrate and wash-water from the uric acid precipitation contains the 
purin bases. This filtrate is alkalinized with ammonia and again precipitated 
with the ammoniacal silver solution. This precipitate is collected on a small 
filter, washed with water, dried and carefully incinerated. The ash is dis- 
solved in nitric acid and the silver chlorid estimated by titration with potassium 
sulphocyanate as described under the determination of chlorids. One c.c. 
of the potassium sulphocyanate solution is equivalent to 0.00734 gram of 
silver. One part of silver, in the form of the silver compounds of the purin 
bases, represents 0.277 gram of nitrogen, or 0.7381 gram of the purin bases. 
It is evident, therefore, that 1 c.c. of the potassium sulphocyanate solution 
will represent 0.002 gram of nitrogen and 0.00542 gram of purin bases accord- 
ing to the following proportion: 

1 : 0.00734 : : 0.277 : x 
x = 0.002 
By multiplying the number of c.c. of potassium sulphocyanate solution used to 
precipitate the chlorids by 0.00542 we obtain the number of grams of purin 
bases in the 200 c.c. of urine originally used. It is wise in determining the 
amount of purin bases to start with a larger quantity of urine, as, for instance, 
600 to 700 c.c, as the amount present in 200 c.c. would be very small. 

This method is apt to give slightly high values for uric acid, as the purin bases 
may not be entirely soluble in the acid solution used to separate them from the uric 
acid. It requires much more equipment than is usually at the disposal of the 
practitioner so that it can hardly be recommended for routine use in general work. 

Method of Rudisch and Kleeberg. 

This method 1 is quite as accurate as is the preceding and has the advantage 
that it can be carried out in from 20 to 30 minutes. The principle is as follows : 
The total purin bodies are precipitated by an excess of silver nitrate, and the 
excess of silver determined volumetrically 2 by potassium iodid, using a mixture 
of nitrous and sulphuric acid with starch solution as an indicator. As the 
silver compounds of the purin bases are soluble in strong ammonia solution, it 
is possible to make a determination of the uric acid and then estimate the purin 
bases by subtraction of this value from the total purin compounds. 

1 Am. Jour. Med. ScL, 1904, CXXVIII, 899. 

2 See Kretschmer, Biochem. Ztschr., 1913, L, 223. 



238 DIAGNOSTIC METHODS. 

Technic. 

One hundred and ten c.c. of urine are treated with 55 c.c. of fiftieth-normal 
AgN0 3 solution and diluted with strong ammonia to 220 c.c. (The fiftieth- 
normal solution of silver nitrate is made by dissolving 3.3932 grams of AgN0 3 , 
which has been heated for 10 minutes with a small amount of water to 120 C, 
in 500 c.c. of water, adding 75 c.c. of strong ammonia and 10 grams of ammo- 
nium chlorid and making the whole up to one liter.) The addition of the 
ammonia dissolves the purin bases, leaving the uric acid in the precipitate. 
The original mixture is now filtered in such a way that two 100 c.c. portions are 
obtained, each portion representing 50 c.c. of urine. With the first of these 
portions an approximate estimation of the uric acid is made, while with the 
second the accurate one is carried out. 

To one of these portions, fiftieth-normal potassium iodid solution is 
added from a buret, a few drops being removed from time to time and added 
to a solution of nitrous-sulphuric acid mixed with a little starch paste. (The 
fiftieth-normal potassium iodid solution should contain 3.32 grams of KI in 
one liter. The nitrous-sulphuric acid mixture is made by mixing 25 c.c. of 
H 2 S0 4 with 75 c.c. of H 2 and 1 c.c. of fuming HN0 3 .) The addition of the 
drops of the solution to the indicator is for the purpose of determining the 
point at which an excess of potassium iodid occurs. This will be shown by 
the appearance of a distinctly blue contact ring. When the solution shows such 
a reaction, on adding a few drops of the mixture to the indicator, the number of 
c.c. of KI is read off, and we are then prepared for the more accurate control. 
It is wise to keep the indicator cold by immersion in ice-water, as otherwise a 
violent reaction may occur. 

The second 100 c.c. portion is then carefully titrated with the fiftieth- 
normal KI solution by running directly about 1 c.c. less of this solution into 
the urine than was required in the first titration. After this point is reached 
the KI solution should be added drop by drop and a test made after the addi- 
tion of each five drops. In this way an accurate end-point may be reached. 
As each 100 c.c. portion represents 50 c.c. of urine it will contain 25 c.c. of 
fiftieth-normal AgN0 3 solution. Subtract from this 25 c.c. the number of c.c. of 
fiftieth-normal KI used and the number of c.c. of silver nitrate which com- 
bined with the uric acid of the urine is obtained. One c.c. of fiftieth-normal 
AgN0 3 solution represents 0.00336 gram of uric acid. By multiplying this 
factor by the number of c.c. of silver nitrate used, we obtain the amount of uric 
acid in 50 c.c. of urine. 

The second part of this method consists in the determination of the total 
purins. One hundred and ten c.c. of urine are treated with 55 c.c. of fiftieth- 
normal AgN0 3 solution and diluted with water to 220 c.c. The remainder of 
the process is exactly as outlined above. The number of c.c. of fiftieth-normal 
silver nitrate used will represent the values for the total purins. 

If the number of c.c. of silver nitrate solution used in the previous deter- 
mination of uric acid be subtracted from the number used in the determination 
of total purin, the result will be the number of c.c. referable to the purin bases. 
One c.c. of fiftieth-normal AgN0 3 solution represents 0.00152 gram of purin 



THE URINE. 



2 39 




bases calculated as xanthin. By multiplying this factor by the number of c.c. 
of AgN0 3 used we obtain the amount of xanthin in 50 c.c. of urine. 

The writer has used this method frequently and has found its results very 
similar to those of the Salkowski-Ludwig method, although the amount of 
uric acid is somewhat less in this method than in the latter one. 

It is very simple, is quick and accurate and may, therefore, be 
recommended for general work. A somewhat similar method 
is advocated by Bartley. 1 The writer finds this latter method 
fairly reliable and simple. 

Ruhemann's Method. 

This method is a very convenient clinical one, although its 
results are by no means as accurate as those of the preceding 
methods. 2 What the general practitioner desires, as a rule, is to 
know whether the uric acid is increased or diminished and does 
not care as to the absolute value. Such results, giving the total 
purins, may be obtained for clinical purposes by this method. 

It consists in the use of a specially graduated tube, the 
uricometer, in which are placed the reagents and the urine to be 
tested (see cut). The calibrations of the tube are such as to 
represent directly the amount of uric acid in parts per 1000. 
The principle of the method is the decolorization of an iodin 
solution by the uric acid of the urine, and the measurement of the 
amount of urine which must be added to a definite amount of 
iodin solution to effect this decolorization. 

Technic. 
Carbon disulphid is placed in the tube up to the mark S, in 
such a way that the lower meniscus of this reagent rests upon 
the mark. A solution of iodin in potassium iodi'd is then added 
so that the upper portion of the meniscus coincides with the mark 
J. This iodin solution has the following composition. 



Iodin, 

Potassium iodid, 
Absolute alcohol, 
Glycerin, 
Distilled water, q.s., 



0.50 gram 
1.25 grams 
7.50 grams 
5.00 grams 
100.00 grams 



The urine is added slowly by means of a pipet until the 
lowest calibration is reached. The glass stopper is inserted and 
the contents of the tube mixed by repeated inversion for about 
15 seconds. The carbon disulphid absorbs the iodin, taking on 
a distinct purple coloration. If this amount of urine does not 
completely decolorize the iodin, shown by the porcelain-like 
color of the carbon disulphid solution, more urine is added and the tube 



Fig. 79.— 
Ruhemann's 
uricometer. 



'Medical Chemistry, Philadelphia, 1904. 

2 See Bradley and Bunta, Jour. Am. Med. Assn. 



19 13, LX, 44. 



240 DIAGNOSTIC METHODS. 

again inverted for 15 seconds. This process is continued until repeated 
shaking of the tube causes the carbon disulphid to assume a pale pink color. 
The reaction is practically ended at this point, as by a little more shaking of the 
contents the indicator will assume the characteristic porcelain-white appear- 
ance. This process requires from 6 to 15 minutes. The amount of uric acid 
is then read off directly from the tube in parts per liter. 

Should the urine contain less uric acid than can be read off from the 
calibrations, a second test is made adding the iodin solution to the mark 
midway between S and J, the amount indicated on the tube being of course 
divided by 2. Conversely, should the urine contain more uric acid than is 
represented by the lower calibration, one adds the iodin solution to the point 
above J and multiplies his reading by 1.5, or adds the iodin solution to the 
second mark above J and multiplies the reading by 2. 

With this method the urine must be acid in reaction. If the urine contains 
a sediment of the urates, it should be thoroughly shaken before being added, so 
that the urates may be in suspension. Any free uric acid which may have sepa- 
rated in the sediment is not determined in this method. Strongly colored 
urines have no influence upon the decolorization. The presence of sugar does 
not interfere with the results, but if albumin be present in large amounts it 
should be removed by acidifying with dilute acetic acid, boiling, and filtering. 
The writer has used this method very frequently and finds it very useful for 
rough estimation of the uric acid outputs. The purinometer, introduced by 
Hall, 1 seems to be a much more reliable and useful instrument, as it employs the 
reagents required by the Salkowski-Ludwig method. 2 

(e). Purin Bases. 

These bodies have been called purin bases, alloxur bases, xanthin bases, 
and nuclein bases. They are of extreme importance from the standpoint of 
physiologic chemistry, but are, clinically, of less value than is uric acid. The 
following bodies have been isolated from the urine in various conditions: 
adenin, guanin, epiguanin, carnin, episarkin, xanthin, hypoxanthin, heter- 
oxanthin, paraxanthin, and methylxanthin. Certain methylated xanthin 
compounds are found in tea and coffee and are, therefore, introduced into the 
system as caffein, theobromin, and theophyllin, being excreted either as xanthin 
or hypoxanthin. Xanthin (C s H 4 N 4 2 ) is 2, 6, dioxypurin. 

These nuclein bases of the urine arise either from the nuclein of the food 
or from the increased nuclein metabolism of the system. Very little is known 
at the present time regarding the absolute variations in the excretion of these 
purin bases in the urine. Salkowski finds an excretion ranging between 0.0286 
and 0.0561 gram (calculated as xanthin), while Camerer regards an average 

*The Purin Bodies, Phila., 1904. 

2 Folin and Macallum (Jour. Biol. Chem., 1912, XIII, 363) have devised a new method 
for uric acid based upon the action of phosphotungstic acid in alkaline solution upon uric 
acid. The depth of color of the blue solution formed is then compared with that of a standard 
solution. Their a uric acid reagent" is prepared by boiling 100 grams of sodium tungstate with 
80 c.c. of 85 per cent, phosphoric acid and 750 c.c. of water for a couple of hours and then dilut- 
ing to 1 liter. See, also, Folin and Denis, Jour. Biol. Chem., 1913, XTV, 95; Autenrieth and 
Funk, Munch. Med. Wchnschr., 1914, LXI, 457; Host, Ztschr. f. klin. Med., 1914, LXXXI, 
113; Benedict and Hitchcock, Jour. Biol. Chem., 1915, XX, 619. 



THE URINE. 24I 

output as 0.087 gram in 24 hours. It is interesting to note that a vegetable 
diet appears to increase this output more than a meat diet, 0.044 gram being 
excreted, according to Camerer, on a meat diet and 0.111 gram on a vegetable 
regime. This finding is the reverse of that for uric acid. As a general rule, it 
may be said that the output of purin bases is from 8 to 10 per cent, of that of uric 
acid, varying from 16 to 60 mg. per diem. It is evident that foods containing 
these substances should be absolutely interdicted in conditions which may be 
traceable to disturbances in the nuclein metabolism. 

An increase in the amount of uric acid is usually associated with an increase 
of the xanthin bases, but at times no such relations obtain, a decrease in these 
bodies being observed. In leukemia an excretion of 0.321 gram has been 
reported by Magnus-Levy. 1 In certain cases of tuberculosis, nephritis, 
epilepsy, migraine, and pneumonia the output may be much increased. Edsall 
finds the urinary output increased as a result of X-ray treatment. Xanthin is 
occasionally found as a constituent of the urinary sediment and may form calculi. 2 

(/). Creatinin (C^NgO). 
The older ideas regarding the excretion of this nitrogenous body have 
suffered a severe shock from the work principally of Folin, Shaffer, Hoogen- 
huyze and Verploegh, Mellanby, and Klercker. Acid urines were supposed to 
contain creatinin and little or no creatin ; while alkaline urines were thought to 
show creatin instead of creatinin. It has been proven, however, that "normal 
fresh urine, whether acid or alkaline, contains creatinin, and if the normal 
subject has not taken creatin in his food during the preceding days his urine 
will not contain creatin, whatever its reaction. There is no normal excretion 
of endogenous creatin, 3 as this, when ingested, is largely retained in the body 
unless the food contains a large amount of protein." Thus Folin has shown 
that the excretion of creatinin on a diet yielding a urinary nitrogen value of 16 
grams, was 1.55 grams or 3.6 per cent, of total nitrogen; while on a nitrogen- 
free diet with a urinary nitrogen of 3.6 grams, the creatinin output was 0.6 
gram or 17.2 per cent, of total N. He showed, further, that while the actual 
amount excreted varied with different individuals, yet for the same person the 
output was practically constant, under the same conditions of health and mus- 
cular activity. He believes, therefore, that creatinin is by far the most re- 
liable index as to the amount of a certain kind of protein metabolism 
occurring daily in any given individual. He bases his conclusions upon the 
facts that "the absolute quantity of creatinin eliminated in the urine on a 
meat-free diet is a constant quantity, differing for different individuals, but 
wholly independent of quantitative changes in the total amount of nitrogen 
eliminated." Shaffer 4 believes that creatinin is not an index of the total 
endogenous protein catabolism, as patients in whom the endogenous catabo- 

1 Loc. cit. 

2 See Hefter, Deutsch. Arch. f. klin. Med., 1913, CIX, 322; also, Koch, Jour. Biol. Chem., 
1013, XV, 43. 

3 This statement does not apply to growing children who show a normal excretion of a 
relatively large amount of creatin. See Mendel and Rose, Jour. Biol. Chem., ion, X, 213, 255 
and 265; also Folin and Denis, Ibid., 19 12, XI, 253. 

4 Am. Jour. Physiol., 1908, XXIII, 1. 

16 



242 DIAGNOSTIC METHODS. 

lism is much increased may excrete very little creatinin. Folin finds that 
the chief factor determining the amount of creatinin eliminated on any special 
diet is the weight of the patient. Fat or corpulent persons yield less creatinin 
per unit of body- weight than do lean ones. It is, therefore, necessary in meta- 
bolic work to consider not only the body- weight but, also, the excess of fat in 
increasing the weight. 

According to Shaffer, the normal excretion of creatinin varies between 
7 and n mg. of creatinin-nitrogen per kilo of body-weight. In pathologic 
subjects it is low, varying from the normal to 2 mg. per kilo of body-weight in 
24 hours. He calls the creatinin-nitrogen excretion per kilo of weight the 
"creatinin coefficient." He thinks that creatinin is an index of some special 
process of normal metabolism taking place largely, if not wholly, in the muscles. 
Upon the intensity of this process appears to depend the muscular efficiency 
of the individual. In acute febrile conditions, in which an increased destruc- 
tion of muscle tissue occurs, an increase is seen in the creatinin output during 
the active febrile period, while in the period of convalescence a diminished 
excretion will be observed. This excretion in fever does not run parallel to the 
muscular efficiency of the individual (Shaffer). Simon has shown that a dimin- 
ished excretion occurs in anemia, marasmus, myositis ossificans, chlorosis, 
phthisis, chronic parenchymatous nephritis, progressive muscular atrophy, 
and pseudohypertrophic paralysis. 

According to Shaffer, creatin may be excreted by subjects of acute fevers, 
in the acute stages of exophthalmic goiter, in other conditions in which there 
is a rapid loss of muscle protein, and by women during the postpartum reso- 
lution of the uterus. Krause 1 has reported the constant presence of creatin 
in diabetes mellitus referable, probably, to a deprivation of carbohydrates. 
Although administration of carbohydrate usually diminishes the creatin excre- 
tion, Folin's theory would indicate that an increased excretion of creatin may 
follow a carbohydrate-rich diet, if the state of nutrition of the body is high, as a 
greater fraction of the creatin of the food would be eliminated. The study of the 
excretion of creatinin and creatin in various conditions is fast becoming of the 
greatest importance. 2 

1 Quart. Jour. Exper. Physiol., 1910, III, 289. 

2 See Mendel, Science, 1909, XXIX, 584; Ditman and Welker, New York Med. Jour., 1909, 
LXXXIX, 1000, 1046 and 1091; Ellis, Jour. Am. Med. Assn., 1911, LVI, 1S70; Skutetzky, 
Deutsch. Arch. f. klin. Med., 1911, CIII, 423; McCrudden, Jour. Exper. Med., 1912, XV, 457; 
Sedgwick, Am. Jour. Dis. Child., 1912, III, 209; Kraus, Arch. Int. Med., 1913, XI, 613; 
Scaffidi, Internat. Beitr. z. Ernahrungsstor., 1913, IV, 401; Orioli, Ibid., 421; Mellanby, 
Proc. Roy. Soc, 1913, LXXXVI, 88; Myers and Volovic, Jour. Biol. Chem., 1913, XIV, 489; 
Myers and Fine, Ibid., 1913, XV, 283 and 305; Ibid., 1913, XVI, 169; Thomas and Goerne, 
Ztschr. f. physiol. Chem., 1914, XCII, 163; Lampert, Ztschr. f. klin. Med., 1914, LXXX, 498; 
Folin and Morris, Jour. Biol. Chem., 1914, XVII, 469; Folin and Denis, Ibid., 493; Benedict, 
Ibid., 1914, XYTII, 183 and 191; Benedict and Osterberg, Ibid., 195; Shaffer, Ibid., 525; 
Tracy and Clark, Ibid., 1914, XIX, 115; Palmer, Means and Gamble, Ibid., 239; Ringer and 
Raiziss, Ibid., 487; Hull, Jour. Am. Chem. Soc, 19 14, XXXYI, 2146; Morris, Jour. Biol. 
Chem., 1915, XXI, 201; Myers amd Fine, Ibid., 377, 3S3 and 389; Janney and Blatherwick, 
Ibid., 567; Myers and Fine, Ibid., 583; Taylor, Ibid., 663; Baumann and Marker, Ibid., 1915, 
XXII, 49; Mac Adam, Biochem. Jour., 19 15, IX, 229; Practitioner, 19 14, XCII, 540; Hutchison, 
Indian Jour. Med. Research, 1915, II, 814; van Hoogenhuyze, Xederl. Tijdschr. v. Geneesk., 
IQ15, I, 1786; Detweiler and Griffith, New York Med. Jour., 1915, CII, 79S. 



THE URINE. 243 

Qualitative Tests for Creatinin. 

Creatinin has the formula, C 4 H 7 N 3 0, with the graphic structure 

HN C = 



HN = C 



H 3 C— N CH 2 

1 %\ The chemical reactions which serve for the detection of creatinin depend 
upon the formation of different colored compounds. It forms a distinct crys- 
talline compound with zinc chlorid, which may be used in the quantitative 
estimation by Salkowski's method. For this process see works on physio- 
logic chemistry. A point to be remembered regarding creatinin is that it re- 
duces copper solutions and may be mistaken for sugar unless care be exercised. 

Weyl's Test. 
To the urine to be tested are added a few drops of a very dilute aqueous so- 
lution of freshly dissolved sodium nitroprussid and a few drops of dilute 
sodium hydrate solution. In the presence of creatinin a ruby-red color appears 
which changes, after a short time, to an intense yellow. If this solution be 
heated with a little glacial acetic acid the color will change to green and finally 
blue. Acetone gives a similar reaction, but on the addition of acetic acid 
changes to a purplish-red instead of green. If the urine be heated previous to the 
application of this test, the acetone may be driven off. This test for creatinin 
is sensitive to 6 parts in 10,000. 

Jaffe's Test. 

To the urine to be tested are added a few drops of a saturated solution of 
picric acid and a few drops of dilute sodium hydrate solution. If creatinin 
be present a red color appears immediately, which increases in intensity and 
remains permanent for a long time. If glacial acetic acid be added the color 
becomes yellow. Acetone gives a reddish-yellow color of less intensity than 
that produced by creatinin. Glucose, if present, may give a red color if the 
mixture be warmed. This test indicates one part of creatinin in 5,000. 

Quantitative Determination. 
Folin's Older Method. 

The principle upon which this determination 1 is based is the comparison 
of the color produced by Jaffe's reaction with that of a standard solution of 
potassium bichromate. A high-grade colorimeter is necessary for this compari- 
son. Folin recommends the use of the Duboscq instrument, while the writer 
finds one made by Sargent & Co., of Chicago, very satisfactory. This latter 
has the advantage of being much less expensive. 

The reagents necessary are (1) a half-normal potassium bichromate solu- 
tion containing 24.55 grams per liter, (2) a saturated picric acid solution contain- 
ing about 12 grams per liter, and (3) a 10 per cent, solution of sodium hydrate. 

1 Am. Jour. Physiol., 1905, XIII, 45. 



244 



DIAGNOSTIC METHODS. 



Technic. 

Ten c.c. of urine are measured into a 500 c.c. volumetric flask, 15 c.c. of 
the picric acid solution and 5 c.c. of the sodium hydrate solution are then added, 
and the mixture allowed to stand for five or six minutes. This interval is used to 
pour a little of the standard bichromate solution into each of the two cylinders of 
the colorimeter. The depth of the solution in one of the cylinders is then 
accurately adjusted to the 8 mm. mark. With the solution in the other cylin- 
der a few preliminary colorimetric readings are made simply for the sake of 
insuring greater accuracy in the subsequent readings of the unknown solution. 
The two bichromate solutions must, of course, be equal in color, and in taking 

their readings no two should differ 
more than 0.1 mm. or 0.2 mm. from 
the true value (8 mm.), leaving out of 
consideration the very first reading 
made, which is sometimes less ac- 
curate. Four or more readings should 
be made in each case, and an average 
taken of all but the first. After a 
while one becomes sure of the true 
point, and can take the average of the 
first two readings. 

At the end of five minutes the con- 
tents in the 500 c.c. flask are diluted 
up to the 500 c.c. mark. The 
bichromate solution is thoroughly 
rinsed out of one of the cylinders by 
means of the unknown solution and 
several colorimetric readings are then 
made at once. The calculation of the 
results is very simple. It is based on 
the experimentally determined fact 
that 10 mg. of perfectly pure creatinin 
give, under the conditions of the de- 
termination, 500 c.c. of a solution, 8.1 
mm. of which have exactly the same 
colorimetric value as 8 mm. of a half- 
normal bichromate solution. 1 
If, for example, it is found that it takes 9.5 mm. of the unknown urine- 
picrate solution to equal the 8 mm. of the bichromate, then the 10 c.c. of urine 
contain 

8.1 , • • 

10 x - - = 8.4 + mg. of creatinin. 

9-5 
1 This relationship applies to the Duboscq instrument, which Folin advises. If one uses 
the Sargent or other colorimeter, he must, of course, make his adjustment according to his 
special instrument. It matters little what point is used for the standard solution, providing 
one has tested his bichromate solution against solutions of creatinin treated as above and has 
.obtained his relative values. 




Fig. 80. — Sargent's colorimeter. (Courtesy 
of E. H Sargent & Co.) 



THE URINE. 245 

If the 10 c.c. of urine used in the test are found to contain more than 
15 mg. or less than 5 mg.of creatinin, the determination should be repeated with 
a correspondingly different amount of urine, because outside of these limits 
the determination is much less accurate. The color of the urine does not 
materially affect the results on account of the great dilution. Sugar and albu- 
min do not interfere with the determination, while acetone and diacetic acid do 
unless one allows a few minutes before reading. 1 

Folin's Newer Method. 

In order to enlarge the scope of the colorimetric method for the determina- 
tion of creatinin and creatin, Folin advises the replacing of the bichromate solu- 
tion with a standard creatinin solution for the color comparison. While crea- 
tinin may be obtained on the market it is usually far from pure, so that one 
should prepare his own creatinin according to the method of Folin 2 or of 
Benedict. 3 

The method is as follows: One c.c. of a standard solution of creatinin (1 
gram per liter, hence 1 c.c. containing 1 mg.) is measured into a 100 c.c. volu- 
metric flask and 1 c.c. of the urine into a similar flask by means of the accurate 
Ostwald pipets (care being taken to allow the pipet to drain against the neck of 
the flask for a few seconds and then to blow out the pipet clean). Twenty c.c. 
of saturated picric acid solution (measured with a cylinder) are added to each flask 
and then 1.5 c.c. of 10 per cent, solution of sodium hydrate are introduced from 
a buret. At the end of 10 minutes the flasks are filled up to the mark with de- 
water, are mixed thoroughly and the relative color of the two solutions tap 
termined in the colorimeter. It makes little difference whether the standard 
solution is set at 10, 15 or 20 mm., as the comparisons are equally exact at any 
one of these points. This standard reading is divided by the reading of the 
unknown to obtain the milligrams of creatinin present in the volume of urine 
(1 c.c.) taken. If the urine reads less than two-thirds or more than one and 
one-half that of the standard the determination should be repeated with more 
or less urine as the case may be. 

Creatin. 

The determination of creatin, as well as of creatinin, is of much importance 
in various conditions. Many methods have been given for such work but the 
following seems to offer the best results with the simplest conditions. 
Folin's Method. 4 

Enough urine to give 0.7 to 1.5 mg. of creatinin is measured into a weighed 
Erlenmeyer Jena flask (capacity 200 c.c). Twenty c.c. of a saturated picric 
solution, about 130 c.c. of water and a few very small beads to promote even 
boiling are added and the mixture is gently boiled, preferably over a micro- 
burner, for about 1 hour. At the end of this time increase the heat and boil 
down the solution to about 20 c.c. Transfer the flask to the scales and add 

1 See Greenwald, Jour. Biol. Chem., 19 13, XIV, 87; also, Thompson, Wallace and Clot- 
worthy, Biochem. Jour., 1913, VII, 445. 

2 Jour. Biol. Chem., 1914, XVII, 463 and 469. 

3 Jour. Biol. Chem., 1914, XVIII, 183. 

4 Jour. Biol. Chem., 1914, XVII, 469. See, also, Benedict, Ibid., 1914, XVIII, 191. 



246 DIAGNOSTIC METHODS. 

enough water to make the total solution equal to 20 to 25 grams. Cool the 
solution in running water, add 1.5 c.c. of 10 per cent, sodium hydrate solution 
and determine the total creatinin as in the above method, using 1 mg. of pure 
creatinin solution as a standard. 

This method gives both the preformed creatinin and that derived from the 
creatin. Subtract the preformed (as determined by the direct method) from the 
total creatinin and obtain the creatinin derived from creatin. Multiply this 
latter figure by 1.16 to determine the actual amount of creatin as such. 

(g). Undetermined Nitrogen. 

By the undetermined nitrogen of the urine is meant the nitrogen remaining 
after that attributable to urea, uric acid, xanthin bases, ammonia, and creatinin 
has been subtracted from the total nitrogenous output. This factor is made 
up of many substances present in variable amount and determined with more 
or less difficulty. 

As the variations in the other nitrogenous constituents have been shown to 
depend to a great extent upon the nature of the diet, so we would expect 
the values for the undetermined nitrogen to show similar fluctuations. Folin 
finds on a diet yielding 16 grams of total nitrogen in the urine an undeter- 
mined nitrogen output of 0.6 gram or 3.75 per cent, of total nitrogen; while on 
a nitrogen-free diet with a urinary nitrogen value of 3.6 grams the undeter- 
mined nitrogen is 0.27 gram or 7.3 per cent. He is led, therefore, to make 
the following generalization: "The absolute quantity of undetermined 
nitrogen decreases under the influence of the starch and cream diet, but in per 
cent, of the total nitrogen there is always an increase." The separation of 
the various factors included in this undetermined nitrogen has clinically little 
value at the present time. 

(1). Amino Acids. 

Theoretically, we should find in the urine, whenever hepatic metabolism 
is disturbed, both mono- and di-amino acids, as these substances are normal 
products of protein hydrolysis in the intestine and are directly converted into 
urea in the normal liver. It has long been known that acute yellow atrophy 
and phosphorus poisoning were associated with a diminished urea output and 
the presence of the two mono-amino acids, leucin and tyrosin, in the urine. 
Recent work 1 with improved methods has shown that the urine contains these 
and other amino acids in any condition in which hepatic activity is impaired, 
so that our old-time diagnostic point of acute yellow atrophy must fall by the 
wayside. Although these substances are found in much larger quantities 
under pathologic conditions, traces of them are present in normal urine, espe- 
cially when the nitrogen intake has been large. Thus von Xoorden regards 
glycocoll (amino-acetic acid) as a normal urinary constituent, its excretion 

1 See Signorelli, Biochem. Ztschr., 1912, XXXIX, 36; Ibid., 1912, XLYII, 482; Galambos 
and Tausz, Ztschr. f. klin. Med., 1913, LXXYII, 14; Damask, Ztschr. f. klin. Med., 1913, 
LXXVTI, 333; Hugounenq, Presse med., 1913, XXI, 825; Van Slvke and Meyer, Jour. Biol. 
Chem., 1913, XVI, 213; and Loffler, Ztschr. f. klin. Med., 1913, LXXVIII, 4S3; Benedict and 
Murlin, Jour. Biol. Chem., 1913, XVI, 385; Labbe and Bith, Rev. de med., 1914, XXXIV, S9; 
Udaondo and Casteigts, Sem. med., 1914, XXI, 741; Geake and Nierenstein, Ztschr. f. physiol. 
Chem., 1914, XCII, 149; Folin, Jour. Am. Med. Assn., 1914, LXEII, 823. 



THE URINE. 247 

averaging 1 gram daily. These acids are also found in cases of gout, pneumonia, 
especially during the absorption of the exudate, in diabetes, and in leukemia. 1 
A large number of these mon-amino acids have been isolated from the 
direct products of protein hydrolysis, but the number found in the urine has 
not been as large owing to the uncertain methods of examination. The intro- 
duction by Fischer of the esterification method has added much to our knowledge 
of these bodies. By the di-amino acids we mean the bodies lysin, 2 arginin and 
histidin, which are collectively known as the hexone bases. 

Benedict and Murlin's Method. 

This method 3 is not as accurate as the gasometric one of Van Slyke, 4 but is 
much more suitable for clinical work. 

Measure into a 500 c.c. Erlenmeyer flask 200 c.c. of a 24-hour specimen of 
urine, which has been diluted to 2000 c.c. Add an equal quantity of a 10 per 
cent, solution of Merck's phosphotungstic acid in 2 per cent, hydrochloric acid. 
Let the mixture stand at least 3 hours, preferably over night. Pour off 250 c.c. 
of the clear supernatant fluid, add 1 c.c. of a 0.5 per cent, alcoholic solution of 
phenolphthalein and then barium hydrate in substance until the whole fluid 
turns decidedly pink (this latter substance should be added a little at a time to 
avoid an excess). Let this mixture stand for 1 hour. Now filter off two 100 c.c 
samples, each representing 50 c.c. of urine. Neutralize these specimens to 
litmus paper by means of N/5 hydrochloric acid and add at once, to each, 10 to 
20 c.c. of formalin which has been previously neutralized with N/10 sodium 
hydrate solution. Titrate the mixture cautiously with N/10 NaOH to a deep 
red color, i.e., until the drop of alkali produces no additional color. Make the 
duplicate determination in the same way and correct the results by deduction of 
the amount of N/10 NaOH necessary to produce the same depth of color in an 
equal quantity of C0 2 -free water with the same quantity of neutral formalin 
added. Each c.c. of N/10 NaOH used in the titration, corrected as above, 
represents 0.0014 gram of nitrogen referable to the amino acids. Multiply the 
nitrogen value by 2 to obtain the percentage, and this latter amount by 20 to 
derive the total amino-acid nitrogen in the 24-hour specimen. 

1 Warfield (Jour. Am. Med. Assn., 1914, LXTI, 436) has attempted to base a diagnostic 
test for pregnancy on the presence in the urine of dialyzable products reacting to ninhydrin. 
This test, however, has no value as the urine, normal or abnormal, always contains dialyzable 
substances, especially amino acids and certain ammonium compounds, which under proper 
conditions will react to riinhydrin. In this connection see Abderhalden's test in the section 
on Blood; also, Jamison, Jour. Am. Med. Assn., 1914, LXII, 1084; Fischer, Ibid., 950 and 
1575; Falls and Welker, Ibid., 1800; Berman, Am. Jour. Obs., 1914, LXX, 192; Holmes, 
Lancet-Clinic, 1914, CXI, 400. 

Malone (Jour. Am. Med. Assn., 1915, LXTV, 1651) , using a modification of the technic of 
Kiutsi (Urine Diagnosis by Means of Filtration Process, Sapporo, Japan, 1914), removes the 
biuret-reacting urinary substances by means of the addition of 0.3 gram of kaolin to 15 c.c. 
of urine, filters this mixture and neutralizes the filtrate This is then submitted to the usual 
Abderhalden technic, employing 10 c.c. of the biuret-free filtrate and 0.2 gram of dried placenta. 
This latter test may prove to be of some diagnostic value. 

2 See Ackermann and Kutscher (Ztschr. f. Biol., 191 1, LVH, 355) who report the presence 
of lysin in a patient with cystinuria. Kocher (Jour. Biol. Chem., 1915, XXII, 295) finds these 
diamino acids largely increased in malignant tissues. 

3 Jour. Biol. Chem., 1913, XVI, 385. 

4 Ibid., 121 and 125. See, also, Chodat and Krummer, Biochem. Ztschr., 1914. LXV, 
392; Harding and MacLean, Jour. Biol. Chem., 1915, XX, 217. 



248 DIAGNOSTIC METHODS. 

(2). Hippuric Acid (C 9 H 9 N0 3 ). 

This acid is a normal constituent of urine and varies between 0.1 and 
1 gram in 24 hours. It is derived to a large extent from foods containing 
benzoic acid, such as prunes, cranberries, green-gages, bilberries and many other 
fruits, and is also formed from the metabolism of tissue and food protein. This 
substance is directly synthesized in the system by the combination of benzoic 
acid with glycocoll, but the exact place of synthesis is uncertain. A pathologic 
increase in the excretion of hippuric acid has been observed in acute febrile 
diseases, marked intestinal putrefaction, hepatic disturbances, and in diabetes 
mellitus, while in cases of acute diffuse and chronic parenchymatous nephritis 
as well as in amyloid kidneys hippuric acid is practically absent from the urine. 1 
(3). Oxyproteic and Alloxyproteic Acids. 

These acids have been isolated from the urine and seem to be constant 
constituents, derived from protein catabolism. Both of them contain sulphur 
and have been credited with forming a large percentage of the neutral sulphur 
output as well as contributing 2 to 3 per cent, of the total N excretion. 2 Bondzyn- 
ski believes the oxyproteic acid accountable for Ehrlich's diazo-reaction (page 
334). Salkowski 3 has shown that a certain amount (3 to 4 per cent.) of the 
urinary nitrogen is precipitable by absolute alcohol and is non-dialyzable. 
This is the so-called " Colloidal N" and is largely referable to oxyproteic acid 
and N-containing carbohydrates. As this factor is increased in many cases of 
carcinoma up to 9 or 10 per cent, of total N, he believes this may be of value in 
diagnosis. However, numerous workers have demonstrated that this increase 
does not always obtain in cases of carcinoma and is found in pregnancy, diabetes, 
some hepatic disorders and in pulmonary tuberculosis. Salomon and Saxl 
have outlined a method for the determination of the sulphur factor of this 
oxyproteic acid as related to total N, and believe that an increase in this factor is 
indicative of carcinoma. 4 

(4). Allantoin (C 4 H 6 N 4 3 ). 
This substance is a product of oxidation of uric acid. It is somewhat 
variable in amount, usually being found only in traces, but after a large intake 

1 Dakin, Jour. Biol. Chem., 1910, VII, 103; Steenbock, Ibid., 1912, XI, 201; Folin and 
Flanders, Ibid., 257; Hryntschak, Biochem. Ztschr., 1912, XLIII, 315; Raiziss and Dubin, Join - . 
Biol. Chem., 1915, XX, 125. 

2 See Browinski and Dombrowski, Ztschr. f. physiol. Chem., 1912, LXXVII, 92; Domansky, 
Monatsschr. f. Psych, u. Neurol., 1912, XXXI, 53; Erben, Prager med. Wchnschr., 1912, 
XXXVII, 427. 

3 Berl. klin. Wchnschr., 1905, XLII, 1581 and 1618; Ibid., 1910, XLVII, 533, 1746 and 
2297. 

4 Marriott and Wolf, Am. Jour. Med. Sc, 1907, CXXXIII, 404;Mancini, Deutsch. Arch, 
f. klin. Med., 1911, CIII, 288; Caforio, Berl. klin. Wchnschr., 1911, XLVIII, 1843; Kojo, 
Ztschr. f. physiol. Chem., 1911, LXXIII, 416; Salomon and Saxl, Wien. klin. Wchnschr., 1911, 
XXIV, 449; Riforma Med., 1911, XXVII, 421; Deutsch. med. Wchnschr., 1912, XXXVIII, 58; 
Neuberg, Der Harn, 1911, 788; Semenow, Folia Urol., 1912, VII, 215; Berl. klin. Wchnschr., 
1913, L, 1436; Greenwald, Arch. Int. Med., 1913, XII, 283; Pribram, Munch, med. Wchn-, 
schr., 1913, LX, 2047; Dozzi, Gazz. d. osp., 1913, XXXIV, 1007; Marenduzzo, Riforma Med., 
1913, XXIX, 1 149; Thar and Beneslawski, Biochem. Ztschr., 1913, LII, 435; Lehmann, 
Deutsch. Arch. f. klin. Med., 1913, CXII, 376; Stadtmuller and Rosenbloom, Arch. Int. Med., 
1913, XII, 276; Sassa, Biochem. Ztschr., 1914, LXIV, 195; de Bloeme, Swart and Terwen, 
Munch, med. Wchnschr., 1914, LXI, 1718; Biochem. Ztschr., 1914, LXV, 345; von Fiirth, 
Ibid., 1915, LXIX, 448; Goodridge and Kahn, iBiochem. Bull., 1915, IV, 118; Damask, 
Wien. klin. Wchnschr., 1915, XXVIII, 499. 



THE URINE. 249 

of meat especially rich in nuclein the amount may be quite perceptible. It is 
found in fairly large quantities in the urine of the new-born, in the amniotic 
fluid, in ascitic fluid, in traces in the urine of pregnancy, and in leukemic 
blood. Pathologic variations have been little studied. 

The best method for the determination of this substance is that of Wiechow- 
ski. 1 A point to be remembered in connection with allantoin is that it may 
be present in the urine in sufficient quantities to reduce copper solutions and 
must, therefore, not be mistaken for sugar. 

(2). Fatty Acids. 

Traces of volatile fatty acids are present in all normal urines. The most 
important of these are formic, 2 acetic, propionic, and butyric acids. They are 
doubtless formed in the intestinal tract by bacterial action upon the carbo- 
hydrates of the food, and may, therefore, be to some extent an index of the 
degree of carbohydrate fermentation. Their normal amount does not much 
exceed 0.01 gram in 24 hours, although Blumenthal 3 gives the figures under 
an ordinary diet as being equivalent to from 50 to 80 c.c. of tenth-normal sul- 
phuric acid (0.25 to 0.39 gram). 

These acids are increased in febrile conditions, the amount running 
parallel to the rise in temperature. This increase in the fatty acids of the 
urine is known as lipaciduria. According to Rosenfeld, this increase in febrile 
states is observed only in those cases in which absorption of decomposing al- 
buminous material occurs, as in all suppurative processes within the system. 
In the convalescent stage of pneumonia these acids may be excreted in increased 
amount, while a diminished output is usually observed preceding the crisis. 
In hyperacidity of the gastric contents the fatty acids of the urine are increased, 
while in hypoacidity they are diminished. In contradistinction to the increase 
of fatty acids in cases of fever associated with suppurative conditions, we find 
a diminution in the amount of fatty acid in scarlet-fever, erysipelas, measles, 
and diphtheria. In cases of acute rheumatism formic acid is said to be excreted 
in large amounts. In cyclic vomiting lactic acid 4 may be increased. 

A simple method of determining the fatty acids of the urine is to acidify 
from 250 to 500 c.c. of urine with 50 to 75 c.c. of dilute sulphuric acid and 
distill. The distillate is then titrated with tenth-normal sodium hydrate 
solution using phenolphthalein as an indicator. The results are expressed in 
terms of the corresponding number of c.c. of tenth-normal acid. 

1 Beitr. z. chem. Physiol, u. Path, 1908, XI, 129; Biochem. Ztschr., 1909, XIX, 378; Ibid., 
1910, XXV, 431. See, also, Plimmer and Skelton, Biochem. Jour., 1914, VIII, 641; Givens, 
Jour. Biol. Chem., 1914, XVIII, 417. 

2 See Dakin and Wakeman, Jour. Biol. Chem., 1911, IX, 329; Steppuhn and Schellback, 
Ztschr. f. physiol. Chem., 1912, LXXX, 274; Dakin, Janney and Wakeman; Jour. Biol. Chem., 
1913, XIV, 341; and Strisower, Biochem. Zstchr., 1913, LIV, 189, for a discussion of formic 
acid as an intermediary metabolic product of both carbohydrates and protein. 

3 Pathologie des Harnes, Berlin, 1903. 

4 For methods of determination see von Fiirth and Charnass, Biochem. Ztschr., 1910, 
XXVI, 199; Mondschein, Ibid., 1912, XLII, 91 and 105; also, Ishihara, Ibid., 1913, L, 468; 
Levene and Meyer (Jour. Biol. Chem., 1913, XV, 65) have shown that lactic acid is a product 
of the intermediary metabolism of carbohydrates. See, also, Yoshikawa, Ztschr. f. physiol. 
Chem., 1913, LXXXVII, 382; von Fiirth, Biochem. Ztschr., 1914, XLIV, 131; Ibid., 156; 
Wien. klin. Wchnschr., 1914, XXVII, 877; Underhill and Steele, Am. Jour. Dis. Child., 1914, 
VIII, 127; Schneyer, Biochem. Ztschr., 1915, LXX, 294. 



250 DIAGNOSTIC METHODS. 

(3). Oxalic Acid. 

The amount of oxalic acid eliminated in the urine in 24 hours varies from 
10 to 20 milligrams. A portion of this excretion is undoubtedly derived from 
the diet, but some of it is produced in the metabolism of the tissues, especially 
of those containing nuclein substances. Although the carbohydrates do not 
take part in the production of oxalic acid normally, we find, in conditions asso- 
ciated with fermentation of the carbohydrates in the stomach, a large increase 
in the output of oxalic acid. As oxalic acid is more or less readily formed 
by oxidation of uric acid, passing through the intermediate stage of oxaluric 
acid, we can readily see that increase of nuclein metabolism may increase the 
output of this substance. Strangely enough, it has been found that the intake 
either of pure nuclein or of nuclein-containing foods was not associated with 
a corresponding increase in the urinary oxalic acid, so that we must assume 
that a large portion of this intake escapes in the feces in the form of calcium 
oxalate. It has, moreover, been found that the administration of oxalates by 
the mouth is not associated with increased excretion either in the urine or feces, 
so that we must assume a decomposition of the oxalic acid into carbon dioxid 
and water somewhere in the system. This conversion appears to take place 
in the intestinal canal under the influence of bacterial action. Crystals of cal- 
cium oxalate are frequently found in the urine in cases showing a marked in- 
crease in the output of ethereal sulphates, so that we must either assume an 
increased formation from the carbohydrates of the food, an increased intake 
and absorption of oxalates of the food, or an increase in the nuclein metabolism 
as a result of absorption of the toxic products of intestinal putrefaction. 1 

Among the foods which are known to contain relatively large amounts 
of oxalic acid we find spinach, rhubarb, tomatoes, carrots, celery, string-beans, 
green peas, potato, figs, plums, strawberries, pepper, cocoa, tea, and coffee. 
Those which contain very little are meat, milk, eggs, butter, cereals, rice, 
asparagus, cucumbers, mushrooms, lettuce, cauliflower, cabbage, pears, 
peaches, grapes, and melons. 

The increased elimination of oxalic acid may or may not be associated 
with a deposition of crystals of calcium oxalate. An increased elimination is 
observed in cases associated with irregular activity of the gastro-intestinal tract. 
These cases usually show many types of nervous disorder, especially neurasthe- 
nia, and are characterized by the large deposit of oxalate crystals in the urine. 
To this condition has been given the name oxaluria, which can hardly be digni- 
fied as a definite pathologic entity. No inference can actually be drawn regard- 
ing the degree of elimination of oxalic acid from the appearance of a deposit 
in the urine, as it has been shown by Flirbringer that the urine may contain a 
large amount of oxalic acid without a sediment of calcium oxalate crystals being 
formed. 2 Such cases, however, should be watched more or less closely as sepa- 
ration of calcium oxalate may occur within the pelvis of the kidney and lead 

1 See Wegrzynowski, Ztschr. f. physiol. Chem., 1913, LXXXIII, 112; also, Aron and 
Franz, Monatsschr. f. Kinderh., 19 14, XII, 645. 

2 Williams and Williams (Arch. Diagnosis, 1913, VI, 263), who show the relationship of 
obscure pain and hematuria to the excretion of small crystals of calcium oxalate. Fittipaldi, 
Rif. Med., 1915, XXX, 981. 



THE URINE. 251 

to the formation of a calculus. In cases of jaundice a marked oxaluria may be 
observed which is directly referable to the associated cholemia. In occasional 
cases of diabetes mellitus the elimination of oxalic acid may be much increased. 

Quantitative Determination. 
Baldwin's Method. 1 

Five hundred c.c. of a mixed 24-hour specimen of urine are treated with 
150 c.c. of 95 per cent, alcohol and the mixture set aside for 48 hours to allow 
the calcium oxalate to precipitate. It is then filtered, each particle of the pre- 
cipitate being transferred to the filter by means of hot water and a rubber- 
tipped glass rod. This precipitate is then washed with hot water and later 
with 1 per cent, acetic acid. The precipitate is then washed from the fil- 
ter-paper by a stream of dilute hydrochloric acid from a wash-bottle until 
every trace of the precipitate is removed from the filter and dissolved. The 
filter is then washed with hot water until the washings are no longer acid in 
reaction. The hydrochloric acid solution and the washings are evaporated 
to about 20 c.c, a little calcium chlorid solution is added, the solution is neu- 
tralized with ammonia, is then rendered slightly acid with acetic acid, and 
95 per cent, alcohol added in an amount equal to one-half the volume of the 
liquid. The mixture is then set aside for 48 hours, after which the precipitate 
of calcium oxalate is collected on an ash-free filter, washed with cold water 
and dilute acetic acid until free from chlorids, and the filter with its contents 
is incinerated in a weighed platinum crucible. This latter process is first 
carried out over a Bunsen burner and later over a blast-lamp. The crucible 
is then dried in a desiccator and weighed. The difference in weight represents 
the amount of calcium oxid obtained from 500 c.c. of urine. Each gram of this 
oxid represents 1.6 grams of oxalic acid. 

(4). Ferments. 

Several ferments 2 have been demonstrated in the normal and pathologic 
urine, but do not seem to have any great clinical importance at the present time. 

Pepsin. 

This ferment is present in practically every specimen of urine. It has 
been found by Grober, Gehrig, Griitzner, Mathes, Stadelmann, and others. It 
seems to be absent or very much diminished in cases of typhoid fever, gastric 
carcinoma, and hypoacidity. 3 In cases of pneumonia, Lenobel and Kun and 
Lochbihler have observed the presence of a ferment, which seemed to be pepsin 
in increased amounts. Scola reports a diminution of the normal pepsin con- 
tent in severe diseases of the nervous system. 

Diastase. 

Traces of this ferment are found in normal urine. It is increased by a 
carbohydrate-rich diet and diminished by a carbohydrate-poor regime. Wohl- 

1 Jour. Exper. Med., 1903, V, 27. 

2 See Lindemann, Ztschr. f. klin. Med., 191 2, LXXV, 58. 

3 See von Kozawa, Internat. Beitr. z. Path. u. Ther., d. Ernahrungsstor., 1912, IV, 44; Fuld 
and Hirayama, Ztschr. f. exp. Path. u. Ther., 1912, X, 248; also, Tachau, Ztschr. f. klin. Med., 
1912, LXXVI, 167; Okada, Mitt, a .d. Med. Fakultat, Tokio, 1914, XI, 293. 



252 DIAGNOSTIC METHODS. 

gemuth 1 believes an increase is associated with a disturbance of pancreatic 
function. It is lessened in nephritis. In diabetes the diastase varies with the 
sugar output. 
Lipase. 
This ferment is present normally only in minute traces. It is found, 
however, in cases of hemorrhagic pancreatitis, jaundice, and in diabetes melli- 
tus. It may be detected by the method of Kastle and Loevenhart which is as 
follows: In each of three flasks are placed 5 c.c. of urine. One of the flasks 
is boiled to destroy the ferment which may be present. To a second flask are 
added a few drops of phenolphthalein solution and the acidity determined 
by titration with tenth-normal sodium hydrate solution. The amount of alkali 
necessary to neutralize the 5 c.c. of urine is then added to each of the other 
flasks. To these flasks are then added 0.25 c.c. of ethyl-butyrate and 0.1 
c.c. of toluol, the flasks being then placed in the incubator at 37 C. for 24 hours. 
To each of these flasks there is then added 1/2 c.c. more tenth-normal hydro- 
chloric acid than the amount of tenth-normal alkali previously added. The 
mixture is then shaken out with 50 c.c. of ether and 25 c.c. of alcohol to remove 
the butyric acid. This is then titrated with tenth-normal sodium hydrate, each 
c.c. of tenth-normal sodium hydrate representing 0.0088 gram of butyric acid. 2 

(5). Mucin-like Substances. 
(a). Mucin. 

True mucin is present in traces in practically all urine. It is found both 
as an insoluble portion which forms the nubecula and as a soluble portion which 
is much smaller in amount than the insoluble form, is precipitated by acetic 
acid, but is easily soluble in a slight excess of the acid. This form of protein is 
derived from the urinary passages and has practically no pathologic importance, 
although it may be much increased in catarrhal conditions of the urinary tract in 
which it may appear as a gelatinous ropy material, or in rare cases in the form 
of casts of the ureter or urethra from 1 to 10 cm. long and 3 to 4 mm. thick. 
These cases are rare and have been reported by von Jaksch under the name 
of " ureteritis membranacea" and by Frank under the name of "pyelitis 
productiva." 

Mucin is precipitated by the ordinary reagents for albumin (to be dis- 
cussed later), but is soluble in an excess of these reagents, so that it need not 
be mistaken for albumin. It is frequently confused with the"nucleo-albumins," 
but may be distinguished, chemically, by the fact that it contains no phosphorus 
and gives on heating with acids a substance which reduces copper solutions. 

(b). Nucleo-albumin. 
The large majority of specimens of urine contain a substance (other than 
true mucin) which is precipitated on the addition of cold acetic acid. Dilute 

1 Biochem. Ztschr., 1909, XXI, 432; Berl. klin. Wchnschr., 1910, XLYII, 92 and 1444. 
See, also, Corbett, Jour. Obst. and Gynec. Brit. Emp., 1913, XXIII, 227; Xeumann, Deutsch. 
Arch. f. klin. Med., 1913, CXI, 164; and Geyelin Arch. Int. Med., 1914, XIII, 96; Marino, 
Semana Med., 1914, XXI, 907; Lister, Brit. Med. Jour., 1914, II, 584; Brown and Smith, Bull. 
Johns Hopkins Hosp., 1914, XXV, 213; King, Am. Jour. Physiol., 1914, XXXY, 301; Kahn and 
Brim, Am. Jour. Obs., 1915, LXXI, 39. 

2 See Quinan, Jour. Med. Research, 1915, XXXII, 45. 



THE URINE. 253 

acetic acid does not redissolve the precipitate, so that true mucin is excluded, 
as the latter is usually dissolved even by dilutions of acetic acid which do not 
precipitate the other bodies. The reaction with acetic acid is somewhat inten- 
sified if the urine be diluted. 

Practically every normal urine contains traces of such substances, which 
will give a precipitate with acetic acid, especially if the salts be removed by 
dialpsis. In all probability this body, found in normal urines, is either euglobu- 
lin or a mixture of this protein with fibrinogen. It has been called "nucleo- 
albumin," but this is in all probability a misnomer. It is probably true that 
real nucleo-albumin is never a normal constituent of the urine. In this con- 
nection it must be stated that much confusion exists regarding the nature of 
true nucleo-albumin. 1 This substance has been considered to be identical with 
nucleo-protein, but with absolutely no basis of chemical facts. Nucleo-protein 
(true nuclein) is a combination of protein with the prosthetic group, nucleinic 
acid, which splits up into phosphoric acid and purin bodies; while nucleo-al- 
bumin (pseudo-nuclein) is a combination of protein with paranucleinic acid, 
which is rich in phosphorus, but does not yield purin bases on hydrolysis. 

Morner 2 believes that most of the so-called "nucleo-albumin" is a com- 
pound of true serum-albumin with an albumin-precipitating body formed on 
addition of acetic acid. He showed that there were three such precipitating 
bodies present in the urine; chondroitin-sulphuric acid 3 was practically always 
present, nucleinic acid occasionally present, and tauro-cholic acid, which may 
be normally present in traces, but in certain pathologic conditions is much in- 
creased. He believes that these precipitating bodies are normally present in 
excess and, therefore, any increase of a precipitate on addition of the acetic 
acid would mean an increased excretion of albumin. The more these precipi- 
tating bodies predominate the more the precipitate resembles "nucleo-albumin." 

From the pathologic standpoint what has been called true "nucleo- 
albumin" appears in conditions destroying the integrity of the epithelium of the 
uriniferous tubules or of the bladder as well as in conditions associated with 
the excretion of pus in the urine. Thus we would expect to find such a body 
in acute nephritis, whether the result of bacterial or of exogenous toxins, in the 
acute febrile diseases, in renal hyperemia, in leukemia, in acute yellow atrophy, 
and in obstructive jaundice, in which case this body is derived doubtless from 
the bile. In cases of nephritis this body, precipitable by acetic acid in dilute 
solution, may precede and follow the true albuminuria. In amyloid kidney 
this body seems to be the chief type of protein present. In orthostatic albumin- 
uria this substance may be the only protein present and may persist after the 
others have cleared up. In cases in which the urine contains a large number 
of epithelial cells, casts, and pus-cells, Matsumoto finds a substance precipi- 
table by acetic acid, but only in very smaD amounts. This finding would seem 
to indicate that nucleo-albumin, if such ever occurs, is at least not an indica- 

1 See Jones, "Nucleic Acids," Longmans Green & Co., New York, 1914. 

2 Skand. Arch. f. Physiol., 1895, VI, 332. 

3 See Levene and La Forge, Jour. Biol. Chem., 1913, XV, 69 and 155; also, Pollitzer, 
Deutsch. Med. Wchnschr., 1912, XXXVIII, 1538; Med. Klin., 1913, IX, 2101; Levene and 
LaForge, Jour. Biol. Chem., 1914, XVIII, 12 3; Ibid., 1915, XX, 433. 



254 DIAGNOSTIC METHODS. 

tion of cellular origin from increased epithelial desquamation. This body 
may be found in general catarrhal conditions of the urinary tract, as in cystitis 
or pyelitis, but in such cases we are more apt to obtain true mucin. 

In the above discussion the writer has not attempted to differentiate these 
bodies, as the reports in the literature have little importance beyond the fact that 
a body precipitable by acetic acid was obtained. Each worker has named this 
body as he understood it and in many cases has had no definite basis for such 
conclusion. To remove it from the urine, add a solution of lead acetate and 
filter; the precipitated phosphates and chlorids carry down this protein. 

(6). Pigments and Chromogens. 
(a). Urochrome. 

This pigment is the chief coloring matter of normal urine, imparting a 
yellow, orange, or a brownish color to the urine, depending upon its concentra- 
tion. It is closely related to urobilin, especially the so-called normal urobilin 
of MacMunn, as this latter body may be readily converted into urochrome by 
evaporation of its aqueous ethereal solution. It is, in all probability, a mixture 
of one or more pigments, contains about 4 per cent, of nitrogen, and is free from 
iron. It is readily soluble in water and alcohol; sparingly soluble in acetic 
ether, amyl alcohol, and acetone; insoluble in ether, chloroform, and benzol. 

An increase of urochromogen, the precursor of this pigment, seems to be of 
some clinical importance. Weisz 1 has introduced the following test for its detec- 
tion. Place the suspected urine in a test-tube and dilute with three times 
its volume of water. Mix well and add 3 drops of a 1 to 1000 aqueous 
solution of potassium permanganate. Shake well. A yellow tint or an increase 
in the yellow tone of the mixture indicates a positive reaction. This reaction 
is an indication of a toxic metabolic disturbance and is given in cases with 
high fever, especially in pulmonary tuberculosis and typhoid fever. It is never 
seen normally and, rarely, in carcinoma. A positive reaction is a contra -indica- 
tion to the use of tuberculin in tubercular cases. 
(b). Uroerythrin. 

This pigment is a constituent of a large majority of normal urines, although 

it is probable that it indicates a slight deviation from the normal. It has been 

called rosacic acid byProut and purpurin by Golding-Bird. To this pigment is 

due the salmon or brick-red color which the urinary sediments take in highly 

concentrated febrile urines. Normally, it may not be present in sufficient 

amount to color the urine, but under pathologic conditions it may impart a deep 

orange tint to this fluid. It is soluble in amyl alcohol, slightly soluble in acetic 

ether and absolute alcohol and very difficultly soluble in water. 

1 Med. Klin., 1910, VI, 1661; Biochem. Ztschr., 1911, XXX, 333; Wien. klin. Wchnschr., 
I9I2,XXV, 1 183. See, also, Heflebower, Am. Jour. Med. Sc, 1912, CXLIII, 2 2i;Paranhos and 
Giolito, Brazil-med., 1913, XXVII, 101; Vitry, Rev. internat. de la tuberc, 1913, XXIII, 325; 
Skorczewski, Ztschr. f. exper. Path. u. Therap., 1913, XIV, 113; Salustri, Policlinico, 1913. 
XX, 1729; Nicola, Gazz. d. osp., 1913, XXXIV, 1465; Keim and Vigot, Presse med., 1914, 
XXII, 153; Martelli and Pizzetti, Pohclinico, 1914, XXI, 182; Gullbring, Hygiea, 1914, LXXVI, 
599; Metzger and Watson, Jour. Am. Med. Assn., 1914, LXII, 1S86; Schaffle, Ibid., 1914, 
LXIII, 1294; Pignacca, Gazz. d. osp., 19 14, XXXV, 353; Tuliato, Ibid., 665; Balduzzi and 
Ballero, Ibid., 2053; Rhein, Munch, med. Wchnschr., 1914, LXI, 2355; Bruni, Gazz. d. osp., 
1915, XXXVI, 401; Ferrannini, Riforma Med., 1915, XXXI, 477; Halbey, Med. Klin., 1915, 
XI, 833; Muhlens, Munch, med. Wchnschr., 1915, LXII, 1067; Pulay, Ibid., 1009. 



THE URINE. 255 

This pigment appears to be increased on a meat diet, after severe exercise, 
profuse perspiration, or by irregular digestion. Pathologically, it is observed 
especially in cases of hepatic insufficiency, in chronic cardiac and pulmonary 
disease, in acute articular rheumatism, in malarial fever, and in general acute 
febrile diseases. In typhoid fever one does not find this pigment as frequently 
as in most other acute febrile conditions. 

(c). Urobilin. 

This substance appears in the urine not as a free pigment, but in the form 
of the chromogen urobilinogen, which is decomposed into urobilin through the 
influence of the light. It is claimed that various types of urobilin are found, as 
for instance the normal urobilin of MacMunn and the pathologic urobilin of 
Jaffe. Whether these are really different bodies is at present an unsettled 
question. Urobilin appears to be identical with the stercobilin of the feces and 
is not the same as the normal fecal hydrobilirubin. Much discussion has 
centered around the origin of this pigment. Passing through the stages of the 
hepatogenous, hematogenous, nephrogenous and histogenetic urobilinuria, 
the general concensus of opinion seems at present to be that most of the urinary 
urobilin is of enterogenous origin. 1 According to this theory, the pathogenesis 
of urobilinuria may be presented as follows: 

"The liver-cell, both in normal and abnormal conditions, forms only 
bilirubin from the blood pigment. Providing there is no marked obstruction 
to the passage of bile into the intestine, the bilirubin is acted upon by bacteria 
which reduce it so completely to urobilin that only traces of bilirubin appear in 
the feces. A part of the urobilin is absorbed and is excreted in the urine, 
while traces appear in the bile and in pathologic transudates. When bacte- 
rial action is excluded, as in the new-born, no urobilin is found in the urine. 
Further, when bile is not present in the intestine, as in cases of absolute oc- 
clusion of the ductus choledochus, urobilinuria does not occur. It is sparingly 
excreted when the production of biliary pigment is diminished, as in hunger, 
while the amount is small or at most normal in cases of incomplete exclusion 
of bile from the intestine. On the other hand, the amount excreted may reach 
abnormal limits if a preceding obstruction, accompanied by stasis, has been 
overcome, and bile flows freely into the intestine. Likewise the quantity may 
be abnormally large if the production of biliary pigment from the red blood- 
corpuscles increases as a result of infection and intoxication, or of hepatic lesions, 
such as cirrhosis and cyanotic induration. In these cases the bile is tenacious 
and the condition may give rise to jaundice although it is seldom that the stasis 
is so great that the bile is completely shut off from the intestine. Indeed, in 
most cases, owing to the excretion of excessive pigments in the bile (pleiochromia), 
a more than normal amount of pigment passes into the intestine, and as a result 
of this there arises a marked urobilinuria with a mild degree of biliary stasis. 
In many cases the stagnation of bile is great enough to cause a passage of 
biliary pigment from the blood into the urine, leading to a marked urobilinuria, 

1 See Wilbur and Addis, Jour. Am. Med. Assn., 1912, LIX, 929; also, Fromholdt and Ner- 
sessoff, Ztschr. f. exper. Path. u. Therap., 1912, XI, 400; Wilbur and Addis, Arch. Int. Med., 
1914, XIII, 235; Feldner, Centralbl. f. d. Grenzgeb. d. Med. u. Chir., 191 5, XIX, 163. 



256 DIAGNOSTIC METHODS. 

a mild degree of bilirubinuria, and a yellow-tinting of the tissues. In other 
cases the absorption of bile is so slight that only yellowing of the tissues results, 
yet the concentration of pigment in the blood does not suffice to permit of its 
excretion by the kidneys, so that marked urobilinuria and yellowing of the tis- 
sues without bilirubinuria ensues" (Weintraud). 

Urobilin is found in febrile conditions, chronic passive congestion, lead- 
poisoning, cases in which extravasation of blood into the tissues occurs, in any 
condition associated with marked hemolysis, hepatic cirrhosis, and in the cases 
of jaundice outlined above by Weintraud. It has been noticed in increased 
amounts in Addison's disease, extrauterine pregnancy, hemophilia, and in 
secondary syphilis. 1 

The presence of an increased amount of urobilin usually causes a dark- 
yellow color of the urine, the foam in such cases being colored, more perhaps 
due to the presence of other pigments than to urobilin itself. 

Urobilin is soluble in ethyl alcohol, amyl alcohol, and chloroform, and 
slightly soluble in ether, acetic ether, and in water. If an acid solution of 
urobilin be examined with a spectroscope, it shows a broad absorption band 
to the right of E y the left-border of which reaches nearly to b, while the right 
border encloses F. If the solution be alkaline, the spectrum shows a less broad 
absorption band between E and F enclosing b. This solution is best made as 
follows: Ten to 20 c.c. of the urine are acidulated with a few drops of hydro- 
chloric acid and shaken out with from 6 to 10 c.c. of amyl alcohol, which then 
shows the characteristic spectrum of acid urobilin. If to a small portion of this 
amyl alcohol solution be added a few drops of 1 per cent, solution of zinc chlorid, 
which has been strongly alkalinized with ammonia, a beautiful green fluores- 
cence appears. With Ehrlich's dimethyl-amidobenzaldehyde reaction (p. 336) 
this pigment gives a distinct red color. 

(d). Indican. 2 

In the decomposition of protein occurring in the intestinal canal indol 
(C 8 H 7 N) and skatol (C 9 H 9 N) are found among the products of this bacterial 
cleavage. These substances are absorbed and oxidized in the blood to indoxyl 
(CgHyNO) and skatoxyl (C 9 H 9 NO). These bodies are then conjugated with 
sulphuric acid forming indoxyl and skatoxyl sulphuric acids, after which they 
are excreted in the form of the potassium salt, indoxyl potassium sulphate 
(C 8 H 6 NO— S0 2 — OK) and skatoxyl potassium sulphate (C 9 H 8 NO— S0 2 — OK). 
To the former of these is given the name indican. This chromogen is present 
in much larger amounts than is the skatoxyl compound so that our discussion 
will include principally the former. 

The absolute amount of indican occurring in the urine depends upon 
the amount of decomposition occurring in the intestine. The largest amounts 
are naturally observed in health following a meat diet, while the urine may be 
practically free from indican on a vegetable or a milk diet. The pathologic 

1 See Simon, Med. Klin., 1913, IX, 1164; Hildebrandt, Arch, f . Yerdauungskr., ioi3,XIX, 
442; Kamsarakau, Med. Obozr., 1913, LXXIX, 559; Molnar, VirchoV s Arch. f. path. Anat., 
1913, CCXIII, 433; Hausmann, Ztschr. f. exper. Path. u. Therap., 1913, XIII, 373. 

2 See Baar, Die Indicanurie, Wien, 1912; New York Med. Jour., 1914, XCIX, 669. 






THE URINE. 257 

variations may be summed up as follows. An increased elimination of indican 
is observed in all diseases which are associated with an increased intestinal 
decomposition. 1 This decomposition usually occurs in the large intestine, but 
may at times take place in the small bowel, in which cases the degree of indicanu- 
ria may be greater owing to the increased absorptive power of the small bowel. 
Although many writers state that indicanuria is not seen in cases of simple 
constipation, the writer must take exceptions to these statements as some of his 
most intense and persistent cases of indicanuria have been found in uncompli- 
cated constipation. Secondly, an increased excretion of indican is observed in 
cases showing diminished peristalsis, as, for instance, in ileus and peritonitis. 
These cases frequently show an intense reaction. 2 If the obstruction leading to 
ileus be in the large bowel, indican is either absent or appears much later than 
is the case if the small bowel be involved. Lastly, in any condition associated 
with protein decomposition anywhere in the system, as, for instance, in empyema, 
putrid bronchitis, abscess formation, etc., indican may be much increased. 

The color of the urine is usually normal when voided, although a large 
amount of indican may be present. In some cases oxidation of this chromogen 
has occurred within the system and the urine appears greenish or even blue 
when voided. If the urine be allowed to stand until decomposition occurs, 
a reddish or bluish metallic-like scum may be observed, due to the conversion 
of the indican into indigo-blue. Most of the tests for the presence of indican 
in the urine depend upon the oxidation of the indican, according to the following 
equation (this body having been previously decomposed by concentrated HC1 
into indoxyl and sulphuric acid). 

2C 8 H 7 NO + 20 = C 16 H 10 N 2 O 2 + 2H 2 0. 
Tests for Indican. 
Jaffe's Test. 

A few c.c. of urine are treated with an equal volume of concentrated 
hydrochloric acid, two or three drops of a strong solution of calcium hypochlorite 
(bleaching powder or "chlorid of lime") are added and the contents mixed. 
Two c.c. of chloroform are then added and the tube inverted several times. In 
this process the indican is oxidized to indigo-blue which is taken up by the 
chloroform. The depth of the blue coloration of the chloroform will serve as 
an approximate estimate of the amount of indican present. In absolutely 
normal urine no blue coloration or, at most, a faint bluish tinge is observed. 
Care must be taken in this test to avoid an excess of the hypochlorite, as this 
will convert the indigo-blue into isatin which is distinctly yellow, according 
to the following equation: 

C 16 H 10 N 2 O 2 + O 2 = 2C 8 H 5 N0 2 . 
Obermayer's Test. 

A few c.c. of urine are mixed with an equal volume of Obermayer's reagent 
(a 0.2 per cent, solution of ferric chlorid in concentrated hydrochloric acid) 

l See Morgan, Am. Jour. Med. Sc, 1912, CXLIV, 827; also, Benedict, Arch. Diagnosis, 
1912, V, 159. 

2 Although there appears to be no definite relation of indicanuria and albuminuria, the 
writer has rarely failed to observe large numbers of casts in urines showing marked indican 
reactions. 

17 



258 DIAGNOSTIC METHODS. 

and the solutions mixed by repeatedly inverting the tube. A few c.c. of chloro- 
form are then added and the tube inverted as before. The presence of any 
appreciable amount of indican is indicated by a dark brown to black coloration 
of the urine and the later absorption of this color by the chloroform, which 
becomes a more or less deep shade of blue. If the urine be very dark in color or 
if bile pigment be present, the pigments may be removed by adding a solution of 
lead subacetate and filtering. If potassium iodid be present this test yields a 
red color which will disappear on addition of sodium thiosulphate solution. 
Salicyluric acid gives a violet coloration. If formalin be used as a preservative, 
indican is incapable of detection by any of the tests given. Thymol-containing 
urines give a violet coloration to the chloroform extract. This color is destroyed 
by sodium hydroxide or thiosulphate. 1 Jolles 2 has made this fact the basis of 
a new test for indican in the urine. 

Instead of using the Obermayer reagent as such, the urine may be mixed 
with the hydrochloric acid and one or two drops of a 10 per cent, solution of 
ferric chlorid may then be added to the mixture. This test is a much more 
reliable one than is the Jaffe test, as the latter is very prone to carry the oxi- 
dation to the isatin stage rather than to the indigo-blue phase. 3 
Skatoxyl-potassium Sulphate. 

A certain amount of skatoxyl-potassium sulphate is formed along with the 
indican. In some cases for reasons not well understood, this pigment or one 
closely allied to it appears in the urine in excess of the indican, so that the 
above test may give distinct red colorations of the choloroform instead of the 
usual blue. Just exactly what pigment causes these red variations is doubtful. 
Some of this pigment is always present along with the indican and may be 
extracted either with hot water or with a mixture of alcohol, ether and water. 
To this red pigment has been given the name indigo-red, skatoxyl-red, urorubin, 
urorhodin, and several others. 

If the urine be heated on applying Jaffe's test, a dark red coloration is 
observed, while if Obermayer's test be used the coloration will be of a reddish- 
violet. 

Rosenbach's Test. 

A few c.c. of urine are boiled and concentrated nitric acid added drop by 
drop during the boiling. The urine takes a deep red color and the foam appears 
bluish-red. If the nitric acid be much in excess the urine will assume a yellow- 
ish-red color and the foam a distinctly yellow tint. If sodium hydrate or 
ammonia be now added drop by drop, a bluish-red precipitate is observed which 
is soluble in an excess of the alkali with a brownish-red coloration. This test 
is due to the presence of indigo-red. Its clinical significance is the same as that 
of indican. This pigment may be obtained directly from the urine by neutral- 
izing it with sodium hydrate and shaking out with ether, when the ether will 
take a distinct red color. 

1 Rosenbloom, New York Med. Jour., 1913, XCVIII, 814. 

2 Ztschr. f . physiol. Cnem., 1913, LXXXVII, 310. See, also, Stanford, Ibid., 188 and Ibid., 
1913, LXXXVIII, 47; also, Edes, Arch. Diagnosis, 1913, VI, 152; Rossi, Policlinico, 1014, 
XXI, 297; Cantelli, Gazz. d. osp., 1915, XXXVI, 833; Jolles , Ztschr. f. physiol. Chem., 1915, 
XCIV, 79; Ibid., 1915, XCV, 29. 

3 See Natonek, Zentralbl. f. inn. Med., 1913, XXXIV, 1124. 



THE URINE. 



259 



Quantitative Determination. 
Wang's Method. 

The principle of this method is the decomposition of indican and its 
oxidation to indigo-blue. This compound is then transformed into indigo- 
sulphuric acid, which is directly determined by titration with potassium per- 
manganate solution. 

Technic. 

A preliminary determination of the relative amount of indican is made 
with Obermayer's reagent. If a strong reaction is obtained, from 25 to 100 c.c. 
of urine are used, while if the reaction be slight 200 to 500 c.c. are necessary. 
The urine should be acidified with acetic acid, unless its reaction be already acid. 

Fifty c.c. of urine, or a larger amount if the conditions mentioned above 
obtain, are treated with 5 c.c. of a 20 per cent, solution of lead acetate, or 
one-tenth the volume of urine in case a larger amount of urine be taken. The 
urine is then filtered and a large and accurately measured portion of the nitrate 
is treated, in a separatory funnel, with an equal volume of Obermayer's reagent. 
This mixture is then shaken out with chloroform, using 30 c.c. of this menstruum 
and shaking for one minute. At least four such chloroform extractions should 
be made, more if the chloroform still extracts indigo-blue from the mixture. 
The chloroform extract is placed in a small flask and the chloroform distilled. 
The residue in the flask is dried for a few minutes on the water-bath to remove 
the last traces of the chloroform, and is washed either with hot water or with a 
mixture of equal parts of alcohol, ether, and water. These solvents remove 
the red coloring matter, leaving the indigo-blue undissolved. The extract is 
filtered through a small filter and the indigo-blue completely transferred to the 
filter, after which it is thoroughly washed with hot water. This indigo-blue is 
dissolved on the filter with boiling chloroform, the filtrate being allowed to run 
into the original flask. The chloroform is again distilled and the residue dried 
on the water-bath. This purified indigo blue is dissolved in 10 c.c. of con- 
centrated sulphuric acid and the solution diluted to 100 c.c. with water. 

This solution of indigo-sulphuric acid is then titrated with standard potas- 
sium permanganate solution of such a strength that 1 c.c. will represent approxi- 
mately 0.0062 gram of indigo-blue. This solution of potassium permanganate 
contains about 3 grams of potassium permanganate to the liter. Its titer is 
determined before use by titrating against a solution of pure indigo-blue in 
sulphuric acid. In making this titration the concentrated solution is not used, 
but a dilute one made by diluting 5 c.c. of the stronger permanganate solution 
to 200 c.c. (each c.c. of which will represent 0.000155 gram of indigo-blue). 
In this titration the blue color of the indigo-sulphuric acid does not change to 
any extent on the addition of the first drops of the permanganate solution, but 
gradually turns greenish and then becomes yellowish or entirely colorless. 
The amount of indigo-blue in the urine used is readily ascertained by multiply- 
ing the number of c.c. of permanganate solution used by the amount of indigo- 
blue represented by the titer of the solution (in the writer's laboratory 1 c.c. 
of the diluted solution equals 0.000155 gram of indigo-blue). 

It has been found by Ellinger that only about 87 per cent, of the theoretical 



260 DIAGNOSTIC METHODS. 

yield is obtained by this method, owing, probably, to the simultaneous for- 
mation of isatin from the indigo-blue. The average daily excretion of indican, 
as revealed by this test, ranges between 5 and 20 mg. 

Folin's Method. 

This method has not, as yet, been extended so as to give absolutely quan- 
titative results. Its principle is the comparison of the color given when urine 
is treated with Obermayer's reagent with that of Fehling's solution as a stand- 
ard. To this standard has been given the arbitrary value of 100. 

Exactly one one-hundredth of the 24-hour specimen of urine is taken for 
each determination and treated with an equal volume of Obermayer's re- 
agent. The indigo-blue is then extracted with 5 c.c. of chloroform until all of 
the pigment has been dissolved. With the chloroform solutions are then made 
colorimetric comparisons with Fehling's solution, using either the Duboscq 
or Sargent instrument. Folin finds the average indican excretion, on this basis, 
from a diet of 119 grams of protein to be 77, while on a nitrogen-free diet no 
indican is found. 

(e). Uroroseinogen. 

This chromogen, shown by Herter 1 to be indol-acetic acid, is converted by 
oxidation into the pigment urorosein, which is soluble in ethyl and amyl alcohol 
but insoluble in chloroform, ether and benzol. If the urine stands for some 
time bacterial action produces this pigment, although an alkaline reaction 
may destroy the coloration. Its amyl-alcoholic solution shows a sharp narrow 
absorption band between D and E. 2 Although this pigment appears in normal 
urine only in traces, it may be markedly increased by a strict vegetable diet. 
Pathologically, it appears in cases of severe gastric and intestinal disturbances, 
tuberculosis, pernicious anemia, nephritis, severe chlorosis, diabetes mellitus, 
-carcinoma, osteomalacia, typhoid fever and dementia praecox. 3 It has, there- 
fore, little differential value although it is, often, as significant as indican of ex- 
cessive protein decomposition in the bowel. 

Test for Urorosein (indol-acetic acid): If 5 c.c. of urine be mixed with 
5 c.c. of chemically pure HC1 and a few drops of a 1 per cent, solution of sodium 
nitrite a characteristic rose-red coloration develops, which is due to oxidizing 
rather than nitrifying action. This pigment also gives a red coloration with 
Ehrlich's dimethyl-amidobenzaldehyd reaction. 

(B). Abnormal Composition. 
(1). Proteins. 
There has been much discussion as to whether a true physiologic pro- 
teinuria occurs. Without going into a discussion of the subject the writer 
wishes to state his belief that a true physiologic proteinuria occurs, but that 
such may never be detected by the usual clinical methods of examination. For 
our purposes, therefore, the presence of protein of one type or another in amounts 
amenable to detection by clinical methods must be considered pathological. 

1 Jour. Biol. Chem., 1908, IV, 107, 239 and 253. 

2 See Riesser, Inaug. Dissert., Konigsberg, 191 1. 

• 3 See Ross, Arch. Int. Med., 1913, XII, 112 and 231. 



THE URINE. 26l 

(a). Serum Albumin. 

From the pathologic standpoint, serum albumin is the most important 
protein found in the urine. The amount excreted in 24 hours is variable and 
does not necessarily have any relation to the severity of the kidney lesion 
should one really exist. From 5 to 10 grams of this protein per diem may be 
regarded as a moderate pathologic excretion, while a lesser would be of little 
significance and a greater would be regarded as excessive. As large amounts 
as 40 grams have been observed, but such findings are exceptional. Morner 
considers an excretion of albumin varying from 25 to 75 mg. per liter as a normal 
output, which amount would, however, escape the usual clinical tests. 

As some of the more delicate tests for albumin, as for instance Spiegler's 
reagent, will show traces of albumin in practically every specimen of urine 
examined, we should limit our conception of albuminuria to those cases which 
react with the ordinary tests rather than with the most delicate. According to 
Hofmeister, the standard upon which one bases a judgment as to the presence 
of a pathologic albuminuria is the formation of a distinct albumin ring within 
three minutes after the urine and nitric acid are in contact in Heller's test (see 
below). Moreover, the term albuminuria should be limited to those cases in 
which there is some disturbance of the renal epithelium, especially of the 
glomeruli. This does not exclude those cases of purely functional albuminuria 
in which no distinct lesion of the kidney exists, as these cases are all associated 
with some abnormality of the excreting organ. 

It not infrequently happens that albumin, as found in the urine, is derived 
from some portion of the urinary tract below the kidney, as when inflammatory 
exudates, blood, lymph, spermatic or prostatic fluids, pus and other extraneous 
material are mixed with the urine after it is excreted by the kidney. 1 To such 
cases is given the name false or accidental albuminuria, in contra-distinction to 
the true albuminuria in which the albumin is present when excreted by this 
organ. Before reporting a finding as one of true albuminuria, it is absolutely 
imperative that extraneous albumin be excluded. 

Functional Albuminuria. 

Not infrequently do we find in perfectly healthy individuals a urine which 
is normal in every way with the exception of the presence of albumin, which 
is easily detected by our ordinary methods. This type of albuminuria is 
observed following severe muscular exercise beyond the point to which the 
subject is accustomed. Thus, in raw recruits under the severe forced marches 
during the early days of their service we find this type very prevalent. Leube 
states that 59 per cent, of such soldiers show a temporary albuminuria, which 
disappears after the subject becomes accustomed to the increased exercise. 2 It 
is a common occurrence to find such a functional albuminuria in football 
players, bicycle riders, crew men, and general athletes after every period of 
increased exertion. This question may be summed up by the statement that 

1 See Belfield (Jour. Am. Med. Assn., 1915, LXIV, 2040) for a discussion of vesicular 
albuminuria due to presence of both globulin and albumin derived from the seminal vesicles. 

2 See von Hecker, Berl. klin. Wchnschr., 1913, L, 1848; Bugge, Norsk Mag. f. Laegevi- 
densk., 1913 LXXIV, 1601; Reber and Lauener, Corr.-Bl. f. Schweiz. Aerzte, 1915, XLV, 
949. 



262 DIAGNOSTIC METHODS. 

the excretion of albumin is simply dependent, under these conditions, upon the 
limit of endurance of the subject, practically everyone being able to produce 
an albuminuria if he overdoes in any way. Moreover, cold baths, excessive 
mental labor, and severe emotion may lead to quite an extensive albuminuria, 
especially in an individual with somewhat lessened resistance. According to 
Rem-Picci albuminuria is a constant finding after cold baths, different sub- 
jects reacting differently to the same stimuli. This albuminuria never lasts 
over 24 hours and may be associated with the appearance of casts and blood, 
as may also the type following increased exercise. The colder the bath and the 
longer the immersion, the more rapid the appearance of albumin. 

Another type of functional albuminuria is that following the intake of a 
heavy protein meal. This is known as " alimentary albuminuria." It has 
been supposed to be due to filtration from the blood, the foreign protein being 
absorbed and excreted without intervening hydrolytic cleavage. Uhlenhuth, 1 
Inouye, 2 and Croftan, 3 working with the precipitin test, have found egg albu- 
min in the blood and urine in these cases. Ascoli 4 later demonstrated both 
serum and egg albumin by this test. Wilson 5 using the fixation of complement 
test, found the main element to be of human origin. Recently Wells, 6 applying 
the anaphylaxis reaction, 7 has shown that sensitized animals do not react to 
egg albumin but show marked reactions to human protein. Owing to this 
disagreement of the anaphylaxis and precipitin tests, it would seem probable 
that the excreted albumin is derived from the blood rather than directly from 
the food, little if any unaltered food protein ever reaching the urine. 8 

The albuminuria observed in the new-born for the first few days of life, 
aside from the influence of feeding upon foreign proteins, is probably a further 
example of a true functional albuminuria. Likewise, the albuminuria observed 
in pregnancy is usually distinctly functional. About 50 per cent, of pregnant 
women show this albuminuria, little difference being observed between primi- 
parae and multiparas. The kidney undoubtedly shows some functional dis- 
turbance in its attempt to eliminate the toxic products absorbed from the fetus. 9 

According to Senator 10 such albuminurias as outlined above should be 
considered functional when slight in degree, transitory in character, occurring 
after unusual strain, either physical or mental, the subjects showing a further 
negative history after the removal of the direct stimulus. Whether this type is 
to be considered physiologic rests entirely upon the conception of the normal or 

1 Deutsch. med. Wchnschr., 1900, XXVI. 734. 

2 Deutsch. Axch. f. klin. Med., 1903, LXXV, 378. 

3 New York Med. Jour., 1909, LXXXTX, 474 

* Munch, med. Wchnschr., 1903, L, 201 and 1761; Ztschr. f. phvsiol. Chem., 1903, XXIX, 
283. 

5 Jour. Path, and Bacteriol., 1909, XIII, 484. 

6 Jour. Am. Med. Assn., 1909, LIII, 863. 

7 See Schittenhelm and Weichardt, Ztschr. f. exper. Path. u. Therap., 1912, XI, 69; Pearce, 
Jour. Exper. Med., 1912, XVI, 349; also, Schloss, Am. Jour. Dis. Child., 1912, III, 341. 

8 See Van Alstyne and Grant, Jour. Med. Research, 1911, XXV, 399; also, Van Alstyne, 
Arch. Int. Med., 1913, XII, 372; Salus, Biochem. Ztschr., 1914, LX, 1; Cameron and Wells, 
Arch. Int. Med., 1915, XV, 746; Bronfenbrenner, Andrews and Scott, Jour. Am. Med. Assn. 
1915, LXW, 1306. 

9 See Bondi, J. and S., Arch. f. Gynak., 1914, CII, 89. 

10 Erkrankungen der Nieren, Wien, 1902. 



THE URINE. 263 

abnormal nature of the stimuli leading to the excretion of albumin. It would 
seem to the writer that the term " physiologic albuminuria" would better be 
given up or at least used with great caution, as it is difficult to believe that 
the severe strain put upon the system and kidneys, in particular, can be dis- 
tinctly physiologic. 1 He is inclined to reserve this term for the excretion of the 
minimal amounts of albumin, which cannot be detected by the simpler clinical 
tests, and to regard the types of albuminuria, as discussed above, as "func- 
tional" (Pavy), or "constitutional" (Martius). 

A second group of cases showing albuminuria is observed in which the 
excretion of albumin may persist for a period varying from days to months, may 
disappear for a variable space and then return for another indefinite length of 
time. To this type is given the name "intermittent albuminuria." This is 
purely functional, no lesion of the kidney being manifest. The cases usually 
show a history of an acute infection or of an antecedent nephritis as the result 
of such infection. The most frequent cause, however, is an uncompensated 
heart lesion which may or may not be associated with a direct renal lesion. 
Not infrequently we find an hereditary intermittent albuminuria in those with a 
distinct neurotic family history. 

The functional albuminuria may, at times, follow a definite course, disap- 
pearing and reappearing with such regularity that it has been styled "cyclic 
albuminuria." In this form the albumin usually disappears from the urine at 
night and when the patient is flat on his back, but reappears during the day 
or when the subject is erect. The terms "orthostatic, orthotic, or postural 
albuminuria" would, therefore, seem to be more appropriate than the former 
appellation. This type is dependent, according to Erlanger and Hooker, upon 
a lowering of the pulse pressure which constantly occurs when the individual 
changes from the recumbent to the erect position. Jehle and, more re- 
cently, Nothmann believe that this type is due to a lordosis, and style it, 
therefore, lordotic albuminuria. 2 Certain pathologic cases, as beginning 
nephritis, may show a cyclic albuminuria which may extend well into the 
period of recovery. In true cyclic cases the negative physical findings 
would lead us to class the condition among the functional albuminurias. 
If a case of this type becomes persistent and casts are frequently found, sub- 
sequent cardiovascular changes will usually appear, changing the case into one 
of true nephritis. The albumin in such cases usually appears in the urine 
after rising and reaches a maximum from noon to 3 or 4 o'clock, then gradually 
declines, disappearing from 8 to 10 p. m. If the subject changes his habits of 
life, the cycle of albuminuria will also change. Along with the variations in the 

1 See Wolf, Arch. Diagnosis, 1912, V, 260. 

2 See Hamburger, Wien. klin. Wchnschr., 1912, XXV, 262; also Frankel, (Deutsch. med. 
Wchnschr., 1912, XXXVIII, 1985), who finds the urinary acidity increased in these cases and 
states that, possibly, the albuminuria is due to abnormal production of acid as Fischer's theory 
assumes. See, also, Gomolitsky, Ztschr. f. klin. Med., 1913, LXXVII, 96; Reyher (Monatsschr. 
f. Kinderhkde., 1913, XII, 82), Dietl (Wien. klin. Wchnschr., 1913, XXVI, 258) and von Jagic 
(Ibid., 1556) believe that tuberculosis is the important factor in this condition. See, also, 
Wendenburg, Arch. f. Kinderhkde., 1913, LXII, 34; Jehle, Die Albuminuric, Berlin, 1914; 
Reyher, Pediatria, 1914, VI, 1; Steensma, Nederl. Tijdschr. v. Geneesk., 1914, LVIII, 248; 
Groenendijk, Ibid., 885; Barlocco, Gazz. d. osp., 1914, XXXV, 2021; Fischl and Popper , 
Jahrb. f. Kinderhkde., 191 5, LXXXI. 



264 DIAGNOSTIC METHODS. 

excretion of albumin, the other urinary constituents fluctuate in the same 
manner, the sequence being, according to Teissier, increase in pigments, al- 
bumin, uric acid, and urea. A peculiarity of this type of albuminuria is that 
it may be even diminished by exercise and is less, therefore, after a hard day's 
work, differing from the albuminuria in nephritis or in the cardiac types. 

While there has been much discussion as to whether such cases are not truly 
pathological, most of them at any rate must have a certain insufficiency of the 
renal epithelium. This may be due to changes in the circulation following 
change in posture, but it is rather surprising that increased exercise would not 
work more strongly in this way. Krehl regards these conditions as relatively 
harmless as they do not usually show any subsequent history of nephritis; 
Broadbent does not believe such cases ever develop actual renal disease; while 
Senator insists that most of them are cases of nephritis. The patients showing 
this type of albuminuria are weak anemic individuals about the age of puberty, 
subject to fainting spells, with a heart showing intermittent attacks of dilatation 
and palpitation, and with a probable congenital weakness of the kidney (37.5 
per cent, having a movable kidney). The adults are neurasthenics with distinct 
vasomotor paralysis. 

A type of albuminuria has been observed in some patients with enlarged 
spleen, in which albumin is present when the patient is flat on his back but is 
absent when he is erect. This has been called "hypostatic albuminuria" Its 
pathogenesis is uncertain, but it can have only an indirect relation to the 
enlarged spleen, as many cases of splenic tumor do not show this albuminuria. 

A still further type of functional albuminuria is known as "albuminuria of 
adolescence." This occurs between the ages of 14 and 16, and then disappears. 
It is different from the cyclic type, although this latter occurs in young people. 
The children are usually anemic, have a neurotic family history, have an 
unstable vasomotor system and, possibly, a congenital weakness of the kidney. 
In this class should be included the albuminuria shown by masturbating 
children or after sexual excess at this period. The kidney does not keep pace 
with the physical growth and activity of the system, so that the association 
with the unstable vasomotor system may account for the albuminuria (Emerson). 
According to Sutherland, movable kidney may be accountable for some of these 
cases, as he finds it present in one-third of his cases. 

Febrile Albuminuria. 
During the course of any acute fever, an albuminuria may be observed 
which is not associated with distinct changes in the renal parenchyma and dis- 
appears with the fall of temperature. The amount of albumin excreted may 
be small or great, depending upon the severity of the toxic action of the bacterial 
products. 1 In ordinary cases there is practically no inflammatory condition 
present in the kidney, the albuminuria being due to an ischemia and a later 
hyperemia. In some of the infectious fevers the influence of the toxins is so 
great that a true nephritis originates, as especially noted in scarlet fever and 
diphtheria. Any febrile albuminuria may pass into a true nephritis, so that 

1 See Grafe, Deutsch. Arch. f. klin. Med., 1914, CXVI, 328. 



THE URINE. 265 

the case must be closely watched for the appearance of symptoms indicating 
such a complication. In some cases, an increase in the albuminuria is observed 
during convalescence, when only traces were previously noted. This is known 
as "colliquative albuminuria." 

Traumatic Albuminuria. 

A transitory albuminuria may be observed following injuries to the kidneys 
or even after bimanual palpation of this organ. The albumin and casts may 
persist for a variable period with no other signs of renal involvement. In 
cases of movable kidney, especially during DietPs crises, an albuminuria may be 
noted due to obstruction in the renal circulation. In cases of ureteral stenosis, 
an albuminuria may be observed as the result of the impeded outflow of urine. 
This same type of albuminuria may be seen after blocking of the ureter by a 
calculus, pressure from a tumor, or twisting of the ureter. 1 

Hematogenous Albuminuria. 

By this type of albuminuria we have in mind one in which the albumin 
is excreted as a result of some alteration in the quality and quantity of the 
normal protein of the blood. On the other hand a distinctly hematogenous 
albuminuria may be the result of the excretion of an abnormal protein. This 
type is observed in purpura, scurvy, pernicious anemia, chronic lead or mercury 
poisoning, syphilis, leukemia, jaundice, cachexia, after the inhalation of 
anesthetics, and in diabetes. 
Toxic Albuminuria. 

This type is directly referable to the influence of various toxic agents 
upon the kidneys. The changes in the kidney may be either of a degenerative 
order, leading to a distinct nephritis, or may be purely circulatory. Among the 
substances causing such an albuminuria we find ether, chloroform, mustard, 
cantharides, mercury, lead, arsenic and antimony compounds, oil of turpentine, 
potassium nitrate and chlorate, phosphorus, carbolic acid, salicylic acid, tar 
compounds (aniline derivatives), petroleum and urotropin. 2 

Neurotic Albuminuria. 

A slight transitory albuminuria may be observed in epilepsy (in which 
condition it may not always be found but is present invariably when marked 
cyanosis is seen during the attack), in apoplexy, tetanus, progressive paralysis, 
exophthalmic goiter, mania, delirium tremens, migraine, brain tumor, injuries 
to the head especially affecting the floor of the fourth ventricle, neurasthenia, 
and various psychoses. Not infrequently we find neurotic patients showing 
an albuminuria as the direct result of perverted metabolism and not as the 
consequence of pathologic changes in the nervous system. 

Albuminuria with Definite Renal Lesions. 

In acute nephritis an intense albuminuria is a constant and important 
symptom. The more acute the case the larger will be the amount of albumin, 
the elimination being generally proportionate to the severity of the disease, 

1 See Evans, Wynne and Whipple (Bull. Johns Hopkins Hosp., 19 12, XXIII, 311), who have 
shown that a reflex albuminuria results from irritation of the urinary bladder. 

2 See Cuntz, Munch, med. Wchnschr., 1913, LX, 1656. 



266 DIAGNOSTIC METHODS. 

although some acute cases may show no albuminuria (Herringham 1 ). The 
percentage of albumin varies inversely as the amount of urine, as a rule, so that 
it is much better to excrete a larger amount of urine with a low percentage of 
albumin than a diminished amount of urine with an increased percentage of 
albumin. The absolute quantity of albumin excreted varies from 0.2 to 1 
per cent. It may reach as high as 5 per cent, or higher, in one case of Senator 
being 8 per cent., but this is rare. The total excretion in 24 hours is rarely over 
25 grams. Nephritis of syphilitic origin appears to be associated with the 
largest outputs of albumin. 

In cases of active renal congestion from exposure to cold or through the 
action of drugs, or in chronic passive congestion due to cardiac, pulmonary, 
or hepatic lesions an albuminuria may be observed without any trace of an 
active renal lesion. The albumin, in these cases, is small in amount and runs 
parallel to the quantity of urine, thus differing from the excretion in true nephritis. 

In chronic parenchymatous nephritis the elimination may be relatively 
large, exceeding, in some cases, that of the acute form. 2 In the chronic inter- 
stitial type the albuminuria is very slight, rarely amounting to more than 5 
grams. In this type of nephritis the albumin may be absent at various exami- 
nations, so that frequent investigations of the urine must be made. In amyloid 
kidney, the urine closely resembles that of the interstitial type of nephritis, a 
total absence of albumin being, however, less frequently observed. The 
serum globulin in this type of kidney disease is relatively more increased than 
in any other type of renal disorder, so that the albumin-globulin quotient may 
be of some importance in diagnosis. 

Tests for Serum Albumin. 

This protein is soluble in distilled water and is coagulated by heat, if the 
solution be acid, at a temperature varying between 56 and 8i° C. The tem- 
perature at which any protein coagulates on heating will depend upon the 
amount of salts present. This protein is precipitated by absolute alcohol and 
by salts of the heavy metals, as well as by the ordinary alkaloidal precipitants. 
It is levo-gyrate, its degree being represented by the following formula {a) D = 
— 62. 6°. It is precipitated by concentrated mineral acids, but is dissolved by 
somewhat large excess. With acetic acid the precipitate first formed is readily 
soluble in a slight excess of the acid. With concentrated alkali serum albumin 
forms an alkali-albuminate which is less soluble in water than is albumin, but 
which is soluble in an excess of the alkali. This fact accounts for the spon- 
taneous precipitation of albumin in a concentrated urine which is alkaline in 
reaction. 

Numerous tests have been given for the detection of albumin in the urine. 
The writer cannot attempt to describe all of these, but must select, therefore, 
those which he has found most useful. 

Before any test for albumin may be made, the urine must be absolutely 

1 Trans. Clin. Soc. London, 1901, XXXIV, 34. 

2 See von Hosslin, Deutsch. Arch. f. klin. Med., 1912, CV, 147; Cook, Jour. Am. Med. 
Assn., 1 914, LXII, 684; Williams, 111. Med. Jour., 1915, XXVIII, 186; Maoris, Grece m6d., 
1915, X\II, 17. 



THE URINE. 267 

clear. It is advisable always to use fresh specimens, but if these are not at 
hand methods must be adopted to clear up the urine. In the majority of 
cases fitration through several folds of filter-paper will usually accomplish this. 
If this does not succeed, as it practically never does if the urine be cloudy from 
the presence of bacteria, recourse must be had to precipitating agents which 
will carry down the suspension of bacteria. 1 Such agents are powdered mag- 
nesium oxid or carbonate, silicic acid, or saw-dust. These substances are 
thoroughly mixed with the urine and the mixture then filtered through double 
folds of filter-paper or plugs of asbestos fiber. The addition of lead acetate 
or any of the salts of the heavy metals is inadvisable, as the precipitates 
formed will include a large part of the albumin, the other precipitants not 
affecting the albumin directly. Occasionally the urine may be cleared by 
centrifugation. 

It has been found that the tests for albumin are rendered more distinct 
if the urine be somewhat diluted. Hallauer has shown that the excess of urea 
and phosphates in a concentrated urine interfere to some extent with the 
delicacy of the reactions. As a rule, a 24-hour specimen is examined or a speci- 
men of the urine voided in the morning and that voided at night. The varia- 
tions of the voidings of the different periods of the day are occasionally quite 
marked, the morning specimen frequently showing no albumin while the 
evening specimen may show quite appreciable amounts. 

Heat Test. 

This test is based upon the principle that serum albumin is coagulated 
by heat especially in the presence of acid. One may use either acetic acid or 
nitric acid, but the conditions of this addition are different in each case. If 
acetic acid be added one must be careful lest he add an excess, as the albumin 
precipitate is soluble in a very slight excess of acetic acid; with nitric acid the 
condition is the reverse, care being taken not to add too little else the albumin 
will not be precipitated by the acid. A few drops of dilute acetic acid are all 
that is required while with nitric acid between one-twentieth and one-tenth of 
the volume of the urine must be added (one to two drops of 25 per cent, nitric 
acid per c.c. of urine). 

Technic. 

A test-tube is filled about three-quarters full of the clear neutral or faintly 

acid urine and heated by directing the flame upon the upper portion of the tube, 

the lower portion being held in the hand. If the fluid remains clear and the 

reaction is acid, no albumin is present. If a cloud is noticed it may be rendered 

more distinct by holding the tube against a black back-ground when the upper 

portion will appear more turbid than the lower. This cloud may be due to 

albumin or calcium phosphate, rarely to calcium carbonate. To determine 

which is the cause, acidulate with a few drops of 5 per cent, acetic acid. If 

the urine becomes clear, the precipitate first noticed is calcium phosphate; 

1 Vaughan (Jour. Lab. and Clin. Med., 1915, 1, 55) calls attention to the erroneous practice 
of heating these bacteria-containing urines with alkali and filtering. As the alkali dissolves the 
bacterial proteins, later tests will show the presence of albumin, which was not present in the 
urine as voided. 



268 DIAGNOSTIC METHODS. 

if it remains turbid and even increases in intensity of turbidity the precipitate 
is albumin; while if due to carbonates an effervescence will be observed. It is 
wise to boil the urine after the addition of each drop of acid, so that the danger 
of getting an excess of acid may be more easily avoided. It is to be remembered 
that, if the protein be very slight in amount, and especially if the urine be 
originally alkaline, the protein will remain in solution owing to the formation 
of acid albumin. If not enough acid is added, the precipitate of phosphates 
may not dissolve, while if too much be added the albumin will dissolve. For 
these reasons it is better to add the acid after boiling. In some cases the fresh 
urine is already too acid to permit of coagulation, so that alkali may be added 
to diminish the acidity. 

The presence of "nucleo-albumin" may lead to a wrong interpretation in 
this test. This substance is precipitated in the cold by acetic acid and may 
thus be differentiated. The resinous acids, which are excreted in the urine 
after the intake of such drugs as copaiba, cubebs, and benzoin, are not so apt 
to interfere with this test unless a large excess of acetic acid be used, which is 
never admissible. 

If nitric acid be used in place of acetic acid the urine is boiled as above 
and concentrated nitric acid added to a strongly acid reaction. The nitric acid 
should never be added before boiling the urine nor should the urine be boiled 
after the nitric acid is added, as traces of albumin will be dissolved by the hot 
nitric acid. A flocculent precipitate is indicative of albumin. The phosphates 
and carbonates do not confuse in this reaction as they are readily dissolved. 
The "nucleo-albumin" is also eliminated by this test as it is readily soluble 
in the excess of acid. In this test the urine should be set aside and allowed 
to cool after the boiling is complete, as albumoses, if present, will separate 
out on cooling as a distinct white flocculent precipitate. A precipitate of uric 
acid may also form on cooling, but this is more granular and is usually colored, 
while the albumin precipitate is white unless an admixture of blood be present. 

In case the urine be poor in salts, the tests are improved by the addition 
of a saturated solution of sodium chlorid. The salts hinder to a great extent 
the solution of the albumin by the acids. In such cases, therefore, it is wise to 
acidify strongly the urine with acetic acid and then add one-sixth its volume of a 
saturated solution of sodium chlorid as recommended by Purdy. 1 The urine 
is now boiled as above when a precipitate on heating will indicate albumin. 
The nucleo-albumin reaction is slight, the albumoses appear only on cooling, 
while the resinous acids may be precipitated but are soluble in alcohol, while 
the albumin is rendered more compact by this reagent. 

Heller's Nitric Acid Test. 

This test is, perhaps, more frequently employed than any other of the tests 
for albumin in the urine. It has a very wide field of usefulness, although it is 
not as delicate as some of those to be mentioned. 2 

1 Ulrich recommends the overlaying of the acidulated salt solution with urine as a contact 
test. Roberts uses a saturated solution of magnesium sulphate. 

2 See Cavazanni, Policlinico, 1914, XXI, 577; Cronquist, Nord. med. Archiv., 1914, XLVII, 
1; Morner, Ibid., 191 5, XLVIII, 1. 



THE URINE. 



269 



A few c.c. of concentrated nitric acid are placed in a test-tube and the 
urine to be tested is allowed to run slowly down the side of the tube in such a 
way as to form a distinct layer of urine above the acid. Some workers advise 
the addition of the acid after the urine, but if this is done it is much better 
practice to allow the nitric acid to flow from a pipet introduced to the bottom 
of the tube. The writer is always accustomed to allow the urine to flow upon the 
nitric acid from a long pipet so that the urine does not perceptibly mix with 
the acid, the tube being held at an angle of 45 degrees. Albumin, if present, is 
precipitated at the zone of contact in the form of a white opaque cloud or ring. 
This precipitate is acid-albumin which is insoluble in a slight excess of acid. 
A red or reddish-violet transparent ring is always obtained with normal urine 
owing to the reaction of the urinary pigments with the nitric acid. If the urine 
contains abnormal coloring matters this colored ring may assume various tints. 
Thus if bile be present a play of colors from red to yellow through blue or green 
takes place, the green being the characteristic coloration; if indican be in excess 





Fig. 81. — Conical 
test-glass. 



Fig. 82. — Horisma- 
scope. 



this colored ring may be bluish or even black; while pigments due to drugs will 
give colors ranging from deep red to violet. This colored ring is usually below 
the white opaque ring due to albumin and tends to extend down into the acid 
instead of up into the urine. If much nitrous acid be present in the nitric acid 
effervescence may be observed to such an extent that the ring of albumin may be 
lost. This albumin ring is usually sharply defined and separated both from 
the urine and acid as a white opaque ring, whose breadth will depend upon 
the amount of albumin in the urine. If the tube be allowed to stand for some 
time the ring will lose its distinct outline, a more or less diffuse cloudiness rising 
throughout the urine. 

Albumin is, however, not the only substance to be precipitated by nitric 
acid under the conditions of this test. Thus we find globulin, albumoses, and 
resins precipitated exactly at the line of contact of urine and acids. If the urine 
be heated the albumoses will dissolve while the albumin becomes more compact. 
If this precipitate be due to resins the precipitate will dissolve in alcohol or 



270 DIAGNOSTIC METHODS. 

ether while the albumin will remain unchanged. It is sometimes wise to shake 
out the urine with ether before applying this test. The globulin ring may be 
differentiated from that of albumin only by separating these proteins by the 
method to be discussed under Serum Globulin. Globulin is usually associated 
with albumin, is practically never found by itself, and has practically the same 
clinical significance, so that for clinical purposes the differentiation of these 
two bodies is unnecessary. Weinberger x has recently shown that the addition 
of thymol, as a preservative of the urine, leads to the formation of a grayish- 
white ring just at the junction of the nitric acid and urine when this test is 
applied. "Below the ring there is a greenish zone extending somewhat into 
the acid, above it a reddish somewhat smaller zone." If this substance is 
suspected, the urine should be extracted by agitation with an equal volume of 
petrolic ether. A somewhat similar reaction has been reported by Kenney 2 
in cases in which several drops of formalin were added as a preservative to a 
small amount of urine. 

Besides these rings at the zone of contact, a further white or yellowish 
ring may be observed at this point. This ring is found in urines which are 
especially rich in urea and appears as a distinctly crystalline ring due to the 
formation of urea nitrate. If the urine be previously diluted this ring does not 
appear. If an excess of uric acid be present in the urine, we observe, on 
allowing the tube to stand for a few minutes, a distinct white ring in the urine 
about 1 to 2 cm. above the point of contact of the acid and urine. 

If the mucin-like bodies previously discussed are present in slight excess, 
a diffuse cloud appears throughout the urine if the fluids have been slightly 
shaken or a distinct ring 1 to 2 cm. above the albumin ring may be observed if the 
urine is carefully added to the acid. This ring is seen in practically all urines, 
is never at the point of contact, and does not appear to have any clinical 
significance. 

Instead of performing this test in a test-tube as described above, it may 
be done in a conical wine-glass, as recommended by Simon and Ogden, or may 
be employed as recommended by Boston. This latter worker uses a flat- 
pointed pipet into which is drawn from 1 to 2 inches of the urine to be tested. 
The exterior of the tube is then wiped perfectly dry and the pipet, with its 
upper end closed with the finger, is introduced to the bottom of a bottle con- 
taining pure nitric acid. By lessening the pressure of the finger the acid 
gradually flows up into the pipet forming a distinct line of contact between 
it and the urine. The same points mentioned above obtain with this test. 
This modification is very simple and may be recommended for general use. 
A further method of performing this test is the use of the horismascope (see 
cut). The urine is placed in the larger tube C and the nitric acid allowed to 
flow through the capillary tube A so that a distinct line of contact is observed. 
The use of this instrument frequently brings out much more clearly the albumin 
ring than do the other modifications. 

This test is frequently employed in combination with heat. Two methods 

1 Jour. Am. Med. Assn., 1909, LII, 13 10. 

2 New York Med. Jour., 1904, LXXX, 403. 



THE URINE. 271 

are available, either one of which may be used, although the results are some- 
what different in the two cases. Some workers advise heating the nitric acid 
previous to the addition of the urine. This does not seem wise to the writer, 
as traces of the acid albumin are undoubtedly dissolved by the hot acid and may 
thus escape detection. The writer is accustomed to heat only the upper portion 
of the tube which contains the somewhat diluted urine. In this way the urates 
and albumoses are thrown out of the field of action while there is much less 
danger of traces of albumin being dissolved. If the various points included 
by this test are remembered and each interfering substance removed by proper 
differentiation, this test is practically the most useful and reliable one for 
the detection of albumin in the urine. 
Potassium Ferrocyanid Test. 

A few c.c. of urine are strongly acidified with acetic acid and a few drops 
of a 10 per cent, solution of potassium ferrocyanid are added drop by drop. 
In the presence of albumin a faint turbidity or a flocculent precipitate will 
be observed, depending upon the amount of albumin present. The slow 
addition of the ferrocyanid is necessary, as an excess of this reagent will dissolve 
the precipitate first formed. In this test albumoses and nucleo-albumin are 
both precipitated. The former is dissolved on heating the mixture, while the 
latter is detected by the precipitate on the addition of the acetic acid unless the 
acid be added in excess. The urates do not interfere if the urine is diluted 
previous to making the test. 

This test is recommended by many as a much more delicate one for 
albumin than those previously described. It reacts with a smaller amount of 
albumin, but it does not, in the writer's opinion, give as much general information 
about the urine as does Heller's test. 
Sulpho-salicylic Acid Test. 

This substance may be used either in the form of a 20 per cent, solution 
or in the solid state. If the solution be used it is added to the acidified urine 
in such a way that a distinct line of contact is formed. Albumin will be shown 
by a distinct white ring at the point of contact. It is preferable, however, to 
add to the urine a small fragment of this substance, when in the presence of 
albumin a turbidity or a white flocculent precipitate will be observed, depending 
upon the amount of albumin. The albumoses are also precipitated by this 
reagent, but dissolve on heating. Neither uric acid nor resins react with this 
substance, while the mucin-like substances are not appreciably affected. 

This test, known as Roch's test, is, perhaps, the most convenient one for 
the use of the general practitioner, as the substance may be readily carried in the 
medicine case and added to the urine at the bedside. 1 
Spiegler's Test. 

As the original reagent of Spiegler was found to be of little value in many 
cases, Jolles 2 has modified it with the following composition: 10 grams of mer- 
curic chlorid, 20 grams of citric acid, and 20 grams of sodium chlorid dis- 
solved in 500 c.c. of water. 

x Folin and Denis (Jour. Biol. Chem., 1914, XVIII, 273) advocate this substance as a 
quantitative precipitant of albumin. 

2 Ztschr. f. physiol. Chem., 1912, LXXXI, 205. 



272 DIAGNOSTIC METHODS. 

The urine is acidified with acetic acid to precipitate the nucleo-albumin if 
present. This substance is filtered off and the filtrate superimposed by means 
of a pipet upon a few c.c. of the above reagent. In the presence of albumin a 
distinct white ring appears at the zone of contact. This reagent precipitates 
the albumoses which are soluble on heating. Nucleo-albumin, if present, should 
be removed before applying the test. In case iodids are present, mercuric 
iodid will be precipitated but may be removed by alcohol. 

This test is the most delicate test for albumin. It shows one part of albu- 
min in 350,000 parts of urine. This is almost too delicate for clinical work as 
it will show albumin in practically every specimen of urine. The writer has 
found this test extremely valuable in cases which showed only a faint reaction 
with Heller's test. If such an urine be treated with Spiegler's reagent a much 
more distinct albumin ring will be observed so that all doubt is thus cleared up. 

Many other tests have been advocated, but the writer does not feel that 
they have any advantages over those outlined above. As far as delicacy of 
reaction is concerned Spiegler's reagent is the most delicate, the sulphosalicylic 
acid, heat and acid, ferrocyanid, and Heller's test following in the order 
named. It will thus be seen that Heller's test is the least delicate of any of the 
ones spoken of above, but the writer is accustomed to use it in general work, 
relying upon Spiegler's reagent to settle mooted points of delicacy of reaction. 
As a general working rule it should be said that no one of the less delicate tests 
should be relied upon without being confirmed by some one of the others. 

Quantitative Determination of Albumin. 
Scherer's Method. 
Fifty c.c. of urine are placed in a beaker and heated upon a water-bath. 
Two or three drops of dilute acetic acid are then added and the mixture boiled. 
A flocculent precipitate of albumin should separate out; if not, a drop or two 
more of acid is added until such a precipitate is obtained. The solution is 
then filtered through an ash-free filter which has been previously dried and 
weighed. The filtrate should be tested by Spiegler's reagent to see if any 
albumin has been dissolved. If this shows no albumin, the precipitate may 
then be dried on the filter-paper after being washed with water, alcohol, and 
ether. If the filtrate shows albumin, another test with a fresh 50 c.c. of urine 
must be made. Much time is saved if a larger quantity of urine be originally 
taken, treated as above, and small portions filtered off and tested for albumin. 
The addition of a small amount of a saturated solution of sodium chlorid will 
facilitate the precipitation of the albumin. The precipitate on the filter-paper 
is dried and weighed, the difference in weight between the original dried paper 
and that with albumin representing the amount of albumin in the 50 c.c. of 
urine used. The total amount is determined by a simple calculation. 

Esbach's Method. 

This test is carried out in a standard graduated glass tube, known as an 
albuminometer (see cut). This tube is filled with acidified urine to the point 
U and the Esbach reagent added to the mark R. This reagent consists of a 
solution of 10 grams of picric acid and 20 grams of citric acid in one liter of 



THE URINE. 



2 73 




distilled water. The tube is now closed with the rubber stopper and inverted 
several times in order to mix thoroughly the contents. It is then allowed to 
stand in a test-tube rack for 24 hours, after which the amount of albumin is 
read off, the graduations on the tube representing the number of grams of 
albumin per liter of urine. 

This test has man}' fallacies and can give only a very approximate determina- 
tion of albumin. The precipitate does not settle evenly. If the albumin reads 
more than 4 grams per liter, the urine must be diluted to a specific gravity 
between 1006 and 1008 and must be kept at constant acidity. If the urine con- 
tains less than 1/2 gram of albumin per liter the test is absolutely 
useless. Further the room temperature must be kept constant at 
about 15° C. or errors as high as 100 per cent, may occur. More- 
over, albumoses, uric acid, creatinin, resinous acids, etc., are precipi- 
tated by this reagent. 

Tsuchiya's Method. 

This method 1 is a modification of the above, the reagent being 
a solution of phosphotungstic acid in acidulated alcohol with the 
following composition: 

Phosphotungstic acid, 1 . 5 grams 

Concentrated HC1, 5.0 c.c. 

95 per cent, alcohol, q. s. ad., 100. o c.c. 

This reagent is used with the Esbach tube as in the above test 
or in the Purdy centrifuge tube discussed later, giving far more 
accurate results than the Esbach test. Normal urine gives only a 
faint unreadable precipitate, ^vhile small amounts of albumin as 
well as large ones are completely precipitated by it. 2 This albu- 
minous precipitate settles regularly and quickly, foaming or floating 
of the precipitate being rarely seen. Variations in temperature 
have little influence on this precipitation. It is to be remembered, 
however, that other urinary albuminous substances will be also pre- 
cipitated. Mattice 3 has shown that this method yields fairly accu- 
rate results for comparative purposes, the figures agreeing closely 
with those of the gravimetric methods. He has also demonstrated 
that the modification of this test, as introduced by Goodman and 
Stern, 4 is neither accurate nor satisfactory, largely because the 
point at which albumin is shown by the first turbidity with this 
reagent (and hence the amount of albumin reacting) varies markedly 
with the dilution of the urine. 

The writer advises, therefore, that this method of Tsuchiya be used for 

1 Zentralbl. f . inn. Med., 1908, XXIX, 105. 

2 If large amounts of albumin are present, the urine must be diluted, as complete precipita- 
tion may not occur with the amount of reagent used. The supernatant fluid must be water 
clear before the precipitation can be regarded as complete. 

3 Arch. Int. Med., 1910, V, 313. See, also, Pfeiffer, Berl. klin. Wchnschr., i9i3,XLIX, 114; 
Semionov, Presse mid., 1914, XXII, 579; Kahn and Silberman, Xew York Med. Jour., 1914, 
C, 66;. 

4 Jour. Am. Med. Assn., 1908, LI, 2055. 




Fig. 83.— 
Esbach's al- 
buminometer. 



274 DIAGNOSTIC METHODS. 

reliable comparative clinical results, the gravimetric or Kjeldahl methods 
being applied for scientific purposes. In case the latter method be used, 
multiply the nitrogen values of the precipitated and washed albumin by 6.3 
to obtain the albumin values. 

It is perhaps unnecessary to state at this point that in speaking of per cent. 
of albumin, one should have reference only to the number of grams of albumin 
by weight in 100 c.c. of urine. The urine very rarely contains more than 5 per 
cent, of albumin, although Salkowski has reported a case in which 8 per cent, 
was observed, the albumin separating out as a white amorphous precipitate on 
standing. This fact should be remembered, as the appearance of such a pre- 
cipitate in the untreated urine might be very misleading. 

Purdy's Centrifugal Method. 

To 10 c.c. of the urine placed in a centrifuge tube, 3 c.c. of a 10 
per cent, solution of potassium ferrocyanid and 2 c.c. of 50 per cent, 
acetic acid are added. The reagents and urine are then mixed by placing the 
thumb over the end of the tube and inverting it several times, after which the 
tube is allowed to stand for 10 minutes. It is then placed in a centrifuge the 
radius of which, with its tubes extended, must be 6 3/4 inches. The tubes are 
revolved for exactly three minutes at a uniform speed of 1,500 revolutions per 
minute. The amount of albumin is then read off in bulk percentage, each 
division of the tube representing 1 per cent., as only 10 c.c. of urine are used 
and the divisions represent tenths of a c.c. One per cent, by bulk represents 
0.021 per cent, by weight of albumin. 

This method is very satisfactory, although not absolutely accurate. It is 
difficult to keep a centrifuge running uniformly at the above rate, unless a 
speed indicator be watched during the entire period. The method is more 
exact and more expeditious than the Esbach method and is to be recommended, 
therefore, for clinical estimations of the amount of albumin. 1 
Removal of Albumin. 

It is advisable in many of the general quantitative tests applied to the 
urine that the albumin should be removed if it is present in more than traces. 
Usually this may be done by acidifying with acetic acid and boiling until the 
precipitate is flocculent. The filtrate in such cases will usually be clear and 
contain no albumin. As this test does not eliminate the albumoses and further 
as the boiling acid may hydrolyze a small amount of the albumin into albumoses, 
Hofmeister recommends the following method. Ten c.c. of a 40 per cent, 
solution of sodium acetate and the same amount of 10 per cent, ferric chlorid 
are added to the urine when it will be colored a bright red. The urine is 
neutralized or rendered very faintly acid and is then boiled. The albumin 
separates out along with the basic ferric acetate and is filtered off. This 
method is not applicable if glucose is present. 2 

(b). Serum Globulin. 

Serum globulin is associated in the blood with serum albumin. This 

1 See Strzyzowski, Ztschr. f. physiol. Chem., 1913, LXXXVIII, 25. 

2 See Tracy and Welker (Jour. Biol. Chem., 1915 XXII, 55) for the use of aluminium 
hydroxide cream in removing albumin. 



THE URINE. 275 

protein is not a single body, but is probably a mixture of two forms known as 
euglobulin and pseudo-globulin. These two fractions differ in their precipita- 
tion and solubility constants and must be looked for in works on physiologic 
chemistry. The former of these probably occurs in most urines, constituting a 
large part of what has been called "nucleo-albumin" (see above). 

Serum globulin, in the specific sense, is usually found in the urine in every 
case in which serum albumin is observed. Cases are reported in which each 
one of these protein bodies has appeared separately, but this is not the normal 
finding. Its excretion as compared with that of serum albumin varies from 10 
to 75 per cent, of the total protein. The relation of the albumin to the 
globulin of the blood is as 1.5 is to 1. This relation, known as the 
" albumin quotient," is by no means the same in the urine. In cases of amyloid 
degeneration of the kidney the globulin may be very much increased beyond 
that observed in other chronic affections of the kidney, the albumin quotient 
being usually lower than one. Senator considers this an important point in the 
diagnosis of this condition. In the various types of nephritis we find the 
globulin being greater the more acute the condition, so that in acute diffuse 
nephritis the albumin quotient may be very low, while in chronic parenchymat- 
ous nephritis the quotient ranges from two and five-tenths to five and five-tenths. 
As the nephritis improves the relative amount of globulin diminishes, but 
increases with each acute exacerbation 1 . 

Globulin is insoluble in water, but soluble in dilute solutions of sodium 
chlorid, dilute acids, or alkalies, unless these be exceedingly dilute and their 
action not prolonged. If, therefore, urine containing globulins be highly 
diluted with water the globulin will be precipitated in the form of a distinct 
cloud. The precipitation constants with various inorganic salts must be 
learned from text-books on physiologic chemistry. 

Qualitative Test for Globulin. 

The. urine is rendered alkaline by the addition of a few drops of ammonium 
hydrate and the precipitated phosphates filtered off. To the filtrate is then 
added an equal volume of a saturated solution of ammonium sulphate, the 
mixture is allowed to stand one hour and is then filtered. The albumoses and 
nucleo-albumin may also be precipitated in this way. Ammonium urate 
does not usually separate out in the hour. The precipitate on the filter is then 
washed with a half-saturated solution of ammonium sulphate until the filtrate is 
albumin-free. A distinct precipitate is usually evidence of the presence of 
globulin, as the albumin is not precipitated until the urine is completely satu- 
rated with the ammonium sulphate. In order to eliminate the other factors 
the precipitate is dissolved in water and heated on a water-bath to coagulate the 
proteins. The solution is filtered, the precipitate washed with water and 
heated on a water-bath with a 1 per cent, solution of sodium carbonate. It is 
then filtered and neutralized with acetic acid. If a precipitate occurs it is globulin, 
as the albumoses and nucleo-albumin would not be precipitated by such treatment. 

Paton advises the use of a contact method in detecting globulin. The 

1 Belfield (Jour. Am. Med. Assn., 1915, LXIV, 2040) calls attention to the presence of 
globulin, with or without albumin, in conditions associated with leakage of the seminal vesicles. 



276 DIAGNOSTIC METHODS. 

phosphates are removed as mentioned above and the filtered urine allowed 
to run down the side of a test-tube containing a few c.c. of a saturated solution 
of sodium sulphate. A white ring will indicate the presence of globulin. 

Quantitative Determination. 

The phosphates are removed as above and 100 c.c. of the clear filtered 
urine are treated with an equal volume of a saturated solution of ammonium 
sulphate or directly saturated with magnesium sulphate. The precipitated 
globulin is collected on a dried and weighed filter and washed with a half- 
saturated solution of ammonium sulphate in the former instance or with a 
saturated solution of magnesium sulphate in the latter case. The final washings 
should show no trace of a reaction for albumin. The funnel with the filter- 
paper and its contents are then dried at no° C. The ammonium sulphate 
is washed from the precipitate with hot water and the precipitate is then dried 
with alcohol, ether, and finally at no° C, until the weight becomes constant. 
The difference between the weight of the filter-paper and the filter-paper plus 
the globulin gives the amount of globulin in 100 c.c. of urine. 

(c). Proteoses. 

These are intermediate products of the digestion of protein by ferments, 
by acids, or by bacteria. In the normal digestion we find the products passing 
through the following stages: protein, acid albumin, primary proteoses 
(of which there are two, namely, protalbumose and heteroalbumose), second- 
ary proteoses (the only well-established representative being deuteroalbumose), 
peptone, amino-acids and hexone bases. The proteoses and peptones are 
very soluble diffusible bodies which are not coagulated by heating. The 
primary proteoses are precipitated by half-saturation with ammonium sulphate, 
the secondary proteoses are precipitated only after complete saturation with 
this salt, while the peptones are not precipitable in either one of these ways. 
The primary proteoses are precipitated by nitric acid, thus differing from the 
secondary types which are not so precipitated. In the urine we find repre- 
sentatives of both types of proteoses, while more or less doubt exists as to 
whether true peptone has ever been isolated from the urine. 

Primary Proteoses. 
Bence-Jones , Protein. 

This body was first discovered by Bence- Jones 1 and was regarded as a 
heteroalbumose. Later work by Magnus-Levy 2 has shown that it is in 
all probability a true albumin, as its digestion products include protalbumose 
which could hardly be derived from true heteroalbumose. As the exact 
chemical nature of this body has not been definitely settled, it will be discussed 
under the above heading, although it probably does not properly belong there. 
This body differs from all other types of protein material which occur in the 
urine in its property of precipitating when heated to as low a temperature as 
40 C. and of practically completely dissolving on boiling, to appear again 
on cooling. A second characteristic of this body is the readiness with 

1 Med. and Chir. Trans., 1850, XXXIII; Phil. Trans. Royal Soc, 1848, I, 55. 

2 Ztschr. f. physiol. Chem., 1900, XXX, 200. 



THE URINE. 277 

which it dissolves in dilute ammonia after it has been precipitated with alcohol. 
The excretion of this body in the urine has been called "heteroalbumosuria," 
"myelopathic albumosuria of Bradshaw," "Kahler's disease" and "Bence- 
Jones' albumosuria." 

The amount of this protein body excreted is somewhat variable. In 
Bence-Jones' original case an output of 6.7 per cent, or a total amount of 70 
grams in the 24 hours was observed, while Coriat 1 reports a case in which none 
was found in the urine, although 4 per cent, was present in the pleuritic effusion. 
Between these limits we find the majority of cases showing an output usually 
not over 1 per cent. The literature contains about 35 cases showing the excre- 
tion of this body. The output of this body appears to be constant during the 
day and not affected in any way by the diet. Little is known regarding the 
direct origin of this body. 2 It undoubtedly has some association with the bone- 
marrow, but just what is not clear. "We may imagine, however, that through 
the agency of the cells of the abnormal tissue, that is their products of metabo- 
lism, the normal transformation of the ingested albumin into tissue-albumin is 
impeded, resulting in the production of the substance in question, which is then 
eliminated as foreign matter" (Simon). 

This body is excreted in cases associated with the occurrence of multiple 
myelomata of the bones, especially when these affect the thoracic skeleton. 
It has been observed in a very few cases of leukemia and in a case associated 
with metastatic carcinoma. 3 However, these findings are so rare that the pres- 
ence of this urinary protein may be regarded as practically pathognomonic of 
multiple myelomata. In some cases of this disease the urine does not show the 
Bence-Jones' body, so that a negative finding does not necessarily preclude the 
condition. Ellinger has shown that this disease may take its course without the 
occurrence of local bone symptoms, but maybe associated with a marked anemia. 
It is, therefore, wise in cases of obscure anemia to test the urine repeatedly for 
this body. 

Tests for Bence-Jones' Body. 

The specific reaction for this protein is observed on heating the acidified 
urine very slowly. At a temperature varying from 50 to 6o° a slight cloud 
changing to a marked turbidity and then into a dense cloud will be observed. 
This may be so intense that the urine appears distinctly milky. This turbidity 
may change into a heavy sticky precipitate or coagulum as the temperature 
approaches the boiling-point. When the boiling-point is reached the pre- 
cipitate entirely or partially dissolves, especially if the boiling be continued from 
one to three minutes. The precipitate may not absolutely all dissolve on boil- 
ing, as variations in the acidity of the urine and the amount of mineral salts 
present may affect this process. If the tube be allowed to stand after being 

1 Am. Jour. Med. Sc., 1903, CXXVI, 631. 

2 Rosenbloom (Arch. Int. Med., 1912, IX, 236 and 255) advances the possibility of osseo- 
albumoid as a precursor of this protein. See, also, Cathcart and Henderson, Jour. Path, and 
Bacteriol., 1912, XVII, 238. 

3 Boggs and Guthrie, Am. Jour. Med. Sc, 1912, CXLIV, 803; Bull. Johns Hopkins Hosp., 
1912, XXIII, 353; Ibid., 1913, XXTV, 368; Folin and Denis, Jour. Biol. Chem., 1914, XVIII, 
277; Schiitz, Deutsch. Arch. f. klin. Med., 1914, CXIII, 441; Sexsmith and Klein, Med. 
Record, 1915, LXXXVTII, 600; Schumm and Kimmerle, Ztschr. f . physiol. Chem., 1914, XCII, 1 . 



278 DIAGNOSTIC METHODS. 

boiled the precipitate returns as the fluid cools. None of the other protein 
bodies give this sequence of precipitation, dissolving, and reprecipitation. 
Hugounenq suggests the name " thermolytic albuminuria" for the excretion of 
this body in the urine. 

Upon the addition of concentrated nitric acid, drop by drop, a temporary 
turbidity develops which disappears on shaking, but persists if more acid be 
added. If the mixture be heated the precipitate will dissolve and reappear 
on cooling. This same reaction may be observed on applying any of the tests 
for serum-albumin outlined above. 

This protein is precipitated from its solution by the addition of two volumes 
of a saturated solution of sodium chlorid to urine which has been previously 
acidified with acetic acid. The addition of two volumes of saturated solution 
of ammonium sulphate likewise causes its complete precipitation. It may be 
then washed with alcohol and ether and dried over sulphuric acid. 

Boston 1 has proposed the following test for this body. Fifteen to twenty c.c. 
of filtered urine are placed in a test-tube and mixed with an equal volume of a 
saturated solution of sodium chlorid, the tube being shaken to insure a thorough 
mixing of the fluids. Two or three c.c. of a 30 per cent, solution of sodium 
hydrate are added and the mixture vigorously shaken. The upper one-fourth 
of the mixture is then gradually heated to the boiling-point and a solution of 
10 per cent, lead acetate added drop by drop, the heating being continued after 
each addition. When the drop of lead solution comes in contact with the 
liquid a copious pearly or creamy cloud appears at the surface, becoming less 
dense as the boiling-point is neared; and when ebullition is prolonged for from 
one-half to one minute the upper portion of the liquid shows slight browning, 
which deepens to a dull black color. Standing intensifies the reaction, and if 
this be prolonged for several hours the black precipitate falls through the clear 
stratum of liquid, collecting in the bottom of the tube as a coarsely granular 
pigment. This reaction is based upon the fact that this body contains a large 
proportion of loosely bound sulphur. Lindemann 2 finds that this body really 
contains no more such sulphur than does serum albumin, while Wood could 
obtain no more blackening than with other proteins. This test would seem to 
have little value as it is in no way distinctive for the Bence- Jones' protein. 

The quantitative determination of this body may be made by precipitation 
with two volumes of saturated solution of ammonium sulphate and the washing 
and drying of this precipitate as previously mentioned. The Tsuchiya method 
is useful as an approximate estimation in the absence of albumin. 

Secondary Proteoses. 

Deuteroalbumose is probably the body which has been found in the urine 
in cases in which peptone was reported. This secondary proteose differs in 
its reactions from the primary proteoses and the Bence- Jones' protein. It is 
precipitated only on complete saturation of the urine with ammonium sulphate. 
In performing this test the urine should be made albumin-free, preferably by 
the Hofmeister method previously discussed. Nucleo-albumin may be precipi- 

1 Clinical Diagnosis, Philadelphia, 1905;. 

2 Deutsch. Archiv. f. klin. Med., 1904, LXXXI, 114. 



THE URINE. 



279 



tated by basic lead acetate. Urine containing deuteroalbumose does not 
become cloudy on boiling; does not regularly give Heller's test, but does react 
with the ferrocyanid test when the neutral salts are present in fairly large 
quantities, and reacts in the cold with sulpho-salicylic acid and with Spiegler's 
reagent, but the precipitate dissolves on heating to reappear again on cooling. 
This reaction with the latter reagents in the cold may be distinguished from 
that of albumin by boiling the mixture and filtering while hot. The albumin 
remains on the filter while the albumose is present in the filtrate, appearing as a 
distinct precipitate as the solution cools. 

Tests for Albumoses. 

To a few c.c. of urine, from which albumin and nucleo-albumin have been 
removed as outlined above, add one-fifth its volume of concentrated acetic acid. 
A 10 per cent, solution of phosphotungstic acid is then added, when the urine 
remains clear on standing if albumoses are absent; while a milky turbidity is 
observed in 5 to 10 minutes if these substances are present. If this precipitate 
be filtered off (warming will facilitate the clumping of the precipitate), washed 
with distilled water and then dissolved on the filter with a very dilute solution 
of sodium hydrate, the solution will have a distinctly blue color. This solution 
is warmed until it becomes clear, more sodium hydrate being added if necessary. 
It is then cooled and the biuret test applied by adding a few drops of strong 
sodium hydrate and a few drops of dilute (2 per cent.) copper sulphate solution. 
On warming this mixture a beautiful-red color will be observed. Fittipaldi 1 
advises that 10 c.c. of urine be treated with 60 c.c. of absolute alcohol and 
allowed to stand until the next day. The alcohol is carefully poured off, the 
precipitate is dissolved in the least possible amount of 30 per cent. NaOH and 
this alkaline solution is treated with aiew drops of a freshly prepared ammoniacal 
nickel solution. 2 In the presence of albumose or peptone an orange-red 
coloration appears. This test seems reliable and does not react with albumin. 

Bang's Method. 

Ten c.c. of urine are heated in a test-tube with 8 grams of finely powdered 
ammonium sulphate until the salt has been dissolved. The mixture is boiled 
for a few seconds and is then centrifugalized for one-half to one minute. The 
supernatant fluid is then poured off and the precipitate extracted with 
alcohol to remove urobilin. After pouring off the alcohol the residue is dis- 
solved in a little water, the solution is boiled to remove albumin and filtered. 
The filtrate is shaken out with chloroform to remove any traces of urobilin 
which may have escaped previously. The watery solution is then poured off 
from the chloroform and tested as above for the biuret reaction. 

Clinical Significance. 

Deuteroalbumose may occur in the urine either alone or associated with 
albumin. It is observed in a great variety of conditions, so that distinct types 
of albumosuria may be noted. 

1 Deutsch. med. Wchnschr., 1911, XXXVII, 1890. Gazz. d. osp., 1911, XXXII, 515. 

2 A 5 per cent, solution of nickel sulphate mixed with an equal volume of ammonia water. 



280 DIAGNOSTIC METHODS. 

Large accumulations of pus anywhere in the system lead to the excretion of 
deuteroalbumose as a result of the breaking down of the pus cells and the later 
absorption of the hydrolyzed material. This form, known as pyogenic albumo- 
suria, is observed in pneumonia during the stage of resolution, in gangrenous 
processes anywhere in the system, in empyema, bronchiectasis, abscess for- 
mation, and in epidemic cerebrospinal meningitis. In this latter condition 
the differential diagnosis from a tubercular meningitis often rests on the appear- 
ance of albumose in the urine. 

A hepatogenous form of albumosuria occurs in any condition associated 
with marked disturbance of hepatic function, as for instance in acute yellow 
atrophy, phosphorus poisoning, cirrhosis, carcinoma, and catarrhal jaundice. 
Little is known of the origin of the albumose in this condition. 

An enterogenous type is observed in cases of gastric or intestinal ulcer, 
whether the latter be due to typhoid fever or dysentery, while intestinal tuber- 
culosis is less frequently associated with the appearance of albumose in the 
urine. In these cases the breaking down of the tissues may be responsible 
for increased absorption both of the products of hydrolysis of the tissues and of 
the food. 

An albumosuria of hematogenous origin has been observed in cases of 
scurvy, leukemia, purpura, dermatitis, poisoning with hemolytic agents, preg- 
nancy, especially after the death of the fetus, and in various psychoses, as well 
as in carcinoma affecting any part of the system. The albumosuria in these 
cases is probably referable to the increased lysis of the cells under the influence 
of the exogenous or endogenous toxins. 

A febrile type is observed in practically all fevers, more especially the 
infectious types, such as measles, scarlet fever, diphtheria, acute articular rheu- 
matism, smallpox, and mumps. This is referable both to the influence of the 
toxins of the disease in producing increased protein disintegration as well as to 
the associated septic conditions. 

A large number of other conditions are associated with the appearance of 
albumose in the urine. Such a state is due to the breaking down either of 
tissue or of an exudate, and may, therefore, appear in almost any type of disease. 
In some cases albumose may appear in the urine, following the ingestion of a 
large amount of albumose. This is the digestive or alimentary albumosuria 
and appears to be indicative of an ulcerative condition somewhere along the 
intestinal tract. Pollak 1 shows that this albumosuria may be due, in part at 
least, to pathological conditions of the kidney, and suggests the name renal 
albumosuria for such cases. 

As previously stated, albumose may be associated in the urine with albumin, 
constituting the mixed albuminuria of Senator. In these cases the albumosuria 
may precede the albuminuria, may alternate with it, or continue after it has 
disappeared. This condition is particularly prominent in cases of nephritis, 
especially of the syphilitic type, and should be watched with care. In any case 
of albumosuria it is necessary to exclude contaminations with foreign material, 
especially with spermatic or prostatic secretions. 
1 Ztschr. f. d. ges. exper. Med., 1914, II, 314. 



THE URINE. 28l 

(d). Peptone. 

True peptone rarely if ever appears in the urine. Peptones are the last 
hydrolytic products of protein which give the biuret reaction. This body is 
not precipitated on saturation with ammonium sulphate as are the other types 
of protein material. Ito reports the finding of true peptones in the urine in cases 
of croupous pneumonia, pulmonary tuberculosis, ulcer of the stomach, and in 
women during the puerperal period. In these cases deuteroalbumose was 
also present, so that there is a possibility that the peptone isolated by Ito was 
derived from the urinary albumose. Many reports of peptonuria are found in 
the literature, but the substance dealt with in practically all of these cases was 
probably some type of albumose and not true peptone. 

0). Hemoglobin. 

This body is the normal coloring matter of the blood and is to be regarded 
from the chemical standpoint as a chromoprotein. In the normal metabolism 
disintegration of red blood-corpuscles is constantly occurring, but this is not suf- 
ficient to lead to a hemoglobinemia and a resulting hemoglobinuria. These two 
conditions must go hand in hand, the latter being impossible without the former. 

When the destruction of the red cell becomes so extensive that the accu- 
mulation of the blood pigment in the blood-current is so great that the liver is 
unable to convert it into bilirubin, hemoglobinemia and hemoglobinuria must 
result. 1 While the distinct limit of destruction of cells necessary to produce 
this condition is Dot definitely settled, it may in general be said to occur when 
approximately one-sixtieth of the hemoglobin of the corpuscles is set free. 
The protein really excreted in the urine is not hemoglobin, but methemoglobin, 
so that the term methemoglobinuria would be better used, although a direct 
hemoglobinemia does obtain. 

From what has been said above it is evident that the excretion of this 
protein will inevitably occur after the use of the so-called hemolytic poisons. 
Among these we find ether, chloroform, snake-venom, arseniuretted hydro- 
gen, phosphorus, hydrogen sulphid, toluylendiamin, mushrooms, anilin, lacto- 
phenin, bile salts, chlorates, pyrogallic acid, naphthol, carbolic acid, carbon 
monoxid, and tuberculin. Hemoglobin will appear in the urine in cases of 
poisoning with the above substances only when the hemoglobinemia is extensive. 
In mild cases, the liver will be called upon to form increased biliary pigment 
and the urine will, therefore, contain bile pigments instead of blood pigments. 
Likewise, we find a hemoglobinuria following transfusion of the blood of ani- 
mals into man, after severe burns, exposure to cold, in the course of any of the 
specific infectious diseases, and in malaria 2 and syphilis. The so-called " black- 
water fever" is more probably a malarial hematuria than a hemoglobinuria. 

A paroxysmal type of hemoglobinuria has been occasionally reported in 
the literature. This occurs in typical paroxysmal forms after exposure to 
cold or exertion, and is often preceded by a typical " infectious" onset, such 
as chill, fever, and malaise, along with pain in the lumbar region. The hemo- 

1 See Wilbur and Addis, Arch. Int. Med., 1914, XIII, 235; Addis, Ibid., 1915, XV, 413. 

2 Urriola (Interstate Med. Jour., 19 12, XIX, 74) claims that the black urinary pigment of 
the urine is pathognomonic of malaria. 



282 DIAGNOSTIC METHODS. 

globin may be excreted for several days and then disappear with no untoward 
symptoms. This condition is very rare and its cause uncertain. 1 An epidemic 
hemoglobinuria occurs at times in the new-born and is associated with a distinct 
hemoglobinemia, jaundice, and cyanosis. 

The urine, in cases of hemoglobinuria, may be clear, but is generally turbid, 
and varies in color from a bright red to almost a black. The turbidity gives 
the appearance of a peculiar smoky or hazy urine. The urine must be ex- 
amined when freshly voided, as blood-corpuscles soon disintegrate in the urine, 
giving it the same appearance as noted in hemoglobinuria. The clinical 
significance of hematuria and hemoglobinuria are much different and should 
not be confounded. If the urine be centrifuged the supernatant fluid will be a 
clear blood-colored liquid and the sediment will show none or very few red cells. 

The tests for the presence of blood pigments must be applied in order to 
differentiate this protein from the other types. Naturally, any specimen of 
urine containing hemoglobin will react to the albumin tests previously given so 
that it may be very difficult to determine whether a true albuminuria is coex- 
istent. As a rule, in such conditions there is an associated nephritis so that all 
the findings of this latter condition may obtain. The chemical tests indicate 
the presence of hemoglobin or of any of its derivatives and do not differentiate 
a hemoglobinuria from a hematuria. Microscopic examination for the presence 
of red blood-cells is the only possible way of clearing up such a diagnosis. A 
spectroscopic examination of the urine will differentiate the types of blood 
pigments (see Blood). 

Tests for Hemoglobin and Derivatives. 
Heller's Test. 
A few c.c. of urine are strongly alkalinized with sodium hydrate and 
heated. Either at once or on standing a brownish-red precipitate of the 
phosphates and the carbonates of the alkaline-earths is formed, the color being 
due to the hematin carried down by the phosphates if blood is present. If 
the urine contains a large amount of foreign pigments, this red coloration may 
not be easily noted. In this case filter off the precipitate and dissolve it in 
acetic acid, when the solution becomes red if blood pigment is present, the 
color gradually fading upon exposure to air. If this test be controlled by the 
spectroscopic tests for hematin in alkaline solution it becomes quite reliable 
and very delicate. It indicates one part of oxyhemoglobin in 4,000 of urine. 

Donogany's Test. 
Ten c.c. of urine are treated with 1 c.c. of ammonium sulphid solution 
and 1 c.c. of pyridin. If blood be present, the urine will assume a more 
or less intense orange color, which may be more evident on looking through 
the test-tube lengthwise. In this case the hemoglobin has been converted into 
hemochromogen, which may be recognized by the spectroscopic test. This 

1 According to Cooke (Am. Jour. Med. Sc, 1912, CXLTV, 203), syphilis is the most impor- 
tant, if not the only, etiologic factor in paroxysmal hemoglobinuria. See, however, Lindbom, 
Hygiea, 1913, LXXV, 885; Ztschr. f. klin. Med., 1913, LXXIX, 147; Young, Jour. Am. Med. 
Assn., 1914, LXII, 356; Widal, Abrami and Brissaud, Sem. Med., 1914, XXXIII, 613; Porges 
and Strisower, Deutsch. Arch. f. klin. Med., 1914, CXVII, 13; Dennie and Robertson, Arch. 
Int. Med., 191 5, XVI, 205. 



THE URINE. 283 

test is more delicate than the previous, showing one part of blood to 8,000 of 
urine. Instead of the above tests, which are more directly applied in urine, the 
guaiac and aloin tests as discussed under Feces may be applied. 

The spectrum of the various blood pigments will be discussed later, so that 
the writer need only refer to the section on Blood for this. If the urine contains 
fresh blood the spectrum is that of oxyhemoglobin, while in cases of hemoglo- 
binuria or of hematuria of renal origin the spectrum is that of methemoglobin. 
The urine to be tested spectroscopically should be slightly acid and perfectly 
clear. If a large amount of blood pigment be present, the spectrum will be 
much clearer if the urine be diluted. This dilution should not be carried too 
far, otherwise the absorption lines will not appear. In testing for methemo- 
globin the spectrum of neutral as well as of alkaline methemoglobin should be 
looked for. 

(/). Fibrin. 

The occurrence of this protein in the urine is very rare. As fibrin is derived 
from fibrinogen through the action of the fibrin ferment, the presence of the 
former body presupposes that the latter two substances have been present 
somewhere along the genitourinary tract. This substance is an elastic, grayish, 
stringy material insoluble in water and alcohol. Chemically, fibrin belongs to 
the group of globulins ; it is soluble with difficulty in dilute saline solutions, is 
coagulated by heat, and precipitated either by great dilution with water or by 
saturation with magnesium sulphate. 

This protein may occur in the urine either in the coagulated form or in 
solution. It is found in any condition in which large amounts of blood are 
present in the urine, whether the blood comes from the kidneys or points below. 
It may coagulate immediately after voiding or may occur as preformed clots 
which are formed either in severe inflammations of the pelvis of the kidney, 
of the ureter, bladder, or urethra. It occurs also in cases of chyluria and rarely 
in direct nephritis. 

In some cases the fibrin is in solution, especially in urines containing no 
blood. This fibrin separates out in the form of a coagulum on standing or 
may change the urine into a distinctly gelatinous mass. This so-called " spon- 
taneously coagulable urine" is seen more frequently in cases of chyluria, but 
may be observed in rare cases of nephritis. 

Test for Fibrin. 

The clotted material is filtered off from the urine, is thoroughly washed 
with water and boiled in a 1 per cent, solution of sodium carbonate. On 
cooling, this solution may be tested as outlined under Serum Albumin. 

(2). Carbohydrates. 

Normally the urine contains traces (0.01 to 0.03 per cent.) of carbohydrates 
which are incapable of detection by the ordinary clinical tests. Besides these 
true carbohydrates the urine contains other substances which react, especially 
toward copper solutions, as do the monosaccharides. These latter reducing 
bodies are uric acid, creatinin, conjugated glycuronic acids, and various pig- 
ments, either normal ones excreted in unusual amounts or abnormal ones 



284 DIAGNOSTIC METHODS. 

excreted in usual amounts. The total output of the reducing bodies of the 
normal urine varies between 2 and 3 grams in 24 hours, while the normal true 
carbohydrates of the urine vary from 0.2 to 1 gram per diem. 

(a). Glucose (d-Glucose) (CH 2 0H— (CHOH) 4 — CHO) . 

The normal blood contains about one part per thousand of glucose. 
Whether this sugar is in the free state or in combination with other molecules, 
as for instance as the so-called jecorin of Drechsel, is at present an unsettled 
question; According to Claude Bernard, sugar will appear in the urine when- 
ever more than three parts per thousand are present in the circulating blood. 
This figure is, in view of the recent work of von Noorden, Stern, and Liefmann, 
much too high, as the average finding in their cases was 0.85 part per thousand. 
The excretion of sugar in the urine is known as glycosuria and presupposes 
an excess of sugar in the blood (hyperglycemia). In only one form of glyco- 
suria do we find absence of this hyperglycemia; that is, in conditions in which 
the kidneys become less impervious than normally to the sugar circulating in 
the blood. This type of glycosuria is most frequently observed in cases of 
poisoning with phloridzin and, also, after intravenous injection of large quantities 
of physiologic salt solution (the so-called "salt glycosuria"), and has led to the 
assumption of the clinical entity u renal diabetes tnellitus." The pathology of 
such a condition is little understood, so that we may for the present disregard this 
type and limit our discussion to the glycosuria which inevitably follows a hyper- 
glycemia. 1 

Glycosuria. 

The normal metabolism is such that any excess of carbohydrate food is 
converted, up to a certain point, into glycogen and stored up in the liver. 
Should this ingestion of carbohydrates exceed the functional power of the liver 
to convert it into glycogen, the excess will pass through the hepatic filter into 
the circulating blood, thus causing directly a hyperglycemia. Unless increased 
muscular activity is sufficient to utilize this excess, the kidneys will excrete sugar 
until the normal relations again obtain. This is the purely alimentary type of 
glycosuria and ceases as soon as the intake is diminished. The type of food 
ingested has much to do with the extent of the glycosuria. Under normal 
conditions it matters relatively little how much carbohydrate is ingested in the 
form of starch, as the products of hydrolysis are gradually absorbed and do not 
lead to overactivity of the liver with a resulting hyperglycemia and glycosuria. 
On the other hand, a certain limit, different for each individual, is observed in 
the amount of sugar which may be ingested without causing a glycosuria. For 
this reason Naunyn has regarded alimentary glycosuria as of two distinct types : 
(1) that following the ingestion of starch, which he styles glycosuria ex amylo, 
and (2) that following the ingestion of an excess of sugar, glycosuria e saccharo. 
The amount of starchy or of saccharine food which a person may ingest without 
a glycosuria is known as the assimilation limit or degree of tolerance for such food. 

1 See de Langen, Nederl. Tijdschr. v. Geneesk., 1913, II, 1454; Ferannini, Riforma Med., 
1914, XXX, 232; Landau, Rev. de Med., 1914, XXXIV, 145; Strouse and Beifeld, Jour. Am. 
Med. Assn., 1914, LXII, 1301; Salomon. Deutsch. med. Wchnschr., 1914, XL, 217; Galambos, 
Ibid., 1301; Gram., Hospitalstid., 1915, LVIII, 329. 



THE URINE. 285 

This factor varies for each individual under normal conditions and under 
pathologic influences is dependent upon the state of the intestines, liver, pan- 
creas, muscles, and kidney. A normal person may stand an intake of from 
200 to 300 grams of glucose without excreting more than traces in the urine, 
but in the majority of persons this figure would, perhaps, be found to be more 
nearly 150 grams than 300. It has been found that the administration of as 
small amounts as 50 grams of galactose and lactose was followed by an excretion 
of these sugars in the urine, while maltose, dextrose, levulose, and saccharose 
required much larger intakes. The excreted sugar, following the increased 
ingestion, is in most cases similar to that taken in, although Moritz has shown 
that some of the polysaccharides may be partially hydrolyzed into their mono- 
saccharide components. It has been found by Worm-Muller that a large intake 
of cane-sugar is not followed by a maximum excretion of this sugar, even though 
the assimilation limit has been greatly exceeded. Thus he observes, after an 
intake of 50 grams of cane-sugar, an excretion of 0.1 gram, while after an intake 
of 150 grams the excretion was only 0.85 gram. 

"Alimentary glycosuria occurs in a healthy person only by saturating 
the organism with soluble carbohydrates. Therefore it is absent after admin- 
istering starch, as in this case no more sugar will be absorbed than can be 
metabolized in the body. It is also scanty, or quite absent, if sugar solutions 
be given on a full instead of on an empty stomach. Naunyn has observed, 
regarding alimentary glycosuria, that there is excreted in the urine only that 
sugar which, according to Ginsberg, reaches the general circulation through 
the thoracic duct, thus avoiding the liver. Still other external influences may 
come into play, such as altered capacity of the tissues, especially those of the 
importantly concerned liver. The result of this may be that excessive doses 
of glucose are stored up as glycogen or fat in a given time. For saccharosuria 
and lactosuria the relationships are somewhat different. Here it is very 
obvious that these double sugars, if given in excessive quantities, are not com- 
pletely split up in the intestine or during their passage through the intestinal wall, 
but enter the general circulation as such. The organism, like most of the yeasts, 
cannot decompose these sugars to any extent, so that they leave the body 
with the molecules unaffected" (Magnus-Levy). 

In testing for the pathologic type of alimentary glycosuria, it is customary 
to give 100 grams of cane-sugar or glucose either in the morning on an empty 
stomach or 2 hours after a very light breakfast. The urine of healthy 
persons should remain free from sugar, while a pathologically lowered limit 
of tolerance will be observed by a more or less extensive glycosuria beginning 
in about 1 hour, reaching a maximum within 2 to 4 hours, and lasting 
about 8 hours. If this glycosuria follows the administration of starchy foods, 
the condition is probably a pure diabetic one, while a glycosuria following 
100 grams of grape-sugar does not necessarily indicate a diabetes. It is prob- 
able that cases showing a somewhat low assimilation limit for sugar belong to 
the type of mild diabetes, although many class them in the general category 
of "hepatic insufficiency." That the liver is incapable in such conditions of 
polymerizing the sugar into glycogen cannot be disputed, but the question at 



286 DIAGNOSTIC METHODS. 

issue is, as von Noorden shows, upon what does the insufficiency of the liver 
depend? I quote from the article in his Handbuch der Pathologie des Stoff- 
wechsels as follows: "The cause cannot be overfilling of the glycogen reservoir, 
by which we explain the alimentary glycosuria of the healthy. In most cases 
the subjects are in ill health and their previous diet anything but excessive, so 
that there is little reason to suppose that their glycogen repository was already 
filled to overflowing. There is no more reason for supposing that they have a 
diminished power of utilizing sugar. x In none of the affections in question are 
the processes of oxidation and of energy production decreased; rather is there a 
great increase in oxidation and especially in the combustion of carbohydrate in 
certain of the diseases, such as Graves' disease and high fever, which predispose 
to alimentary glycosuria. Either the liver cells are unable to polymerize all the 
sugar reaching them or else the glycogen formed is too quickly converted into 
sugar, through some increase in the diastatic process. The latter would imply 
that the automatic regulation between sugar combustion and sugar formation is 
no longer so evenly balanced as in health. Physiologically the diastatic process 
is dependent only on the sugar requirements of the body; here it would also be 
controlled by the sugar supply." 

The idea of hepatic insufficiency as a distinct clinical entity responsible 
for the alimentary glycosuria can hardly hold. One should not be satisfied 
with a mere statement that such a condition exists, but should attempt to ex- 
plain why it does obtain. In many conditions of the liver in which an un- 
doubted insufficiency is present, no glycosuria can be caused by administration 
of a fairly large amount of glucose. On the other hand, administration of levu- 
lose in these conditions produces a distinct alimentary levulosuria. To the 
classes of cases which give this levulosuria should be, according to Strauss, 
applied the term "hepatic insufficiency" rather than to those showing an ali- 
mentary glycosuria. (See p. 305.) 

It would lead me too far afield to discuss the various factors which influence 
the appearance of sugar in the urine. 2 In many cases a restriction of the diet 
within the assimilation limit will rid the urine of sugar, while in others a 
constant production of sugar within the system occurs. This endogenous 
production of sugar may again be of several types. Thus we find in nervous 
conditions, especially in affections in the region of the fourth ventricle, an ex- 
cretion of sugar which continues only so long as the glycogen of the body is not 
used up. If no carbohydrates be taken in the food, glycosuria will soon dis- 
appear. This condition is due to interference with the normal nervous con- 
trol of the glycogenic function of the liver. This type of glycosuria, to which 
the name "neurohepatogenous glycosuria" has been applied, is found in a 

1 Minkowski, Luck, Murlin and, more recently, Roily and David assert that this is not true, 
but that the primary factor in human diabetes is an inadequate utilization of carbohydrates 
rather than the overproduction of sugar as von Noorden's theory would demand. 

2 See Barrenscheen, Biochem. Ztschr., 1913, LVTII, 277; Woodyatt, Jour. Biol. Chem., 
1913, XIV, 38; Greer, Witz emann and Woodyatt, Ibid., 1913, XVI, 455; Ringer, Ibid., 1914, 
XVII, 107; Roily and David, Munch, med. Wchnschr., 1914, LXI, 169; Macleod, Jour. Am. 
Med. Assn., 1914, LXII 1222; Sansum and Woodyatt Jour. Biol. Chem., 1914, XVII, 521; 
Greenwald, Ibid., 1914, XWII, 115; Woodyatt, Ibid., 1915, XX, 129; Moorhouse, Patterson 
and Stephenson, Biochem. Jour., 1915, IX, 171. 



THE URINE. 287 

variety of conditions, such as progressive paralysis, multiple sclerosis, cerebral 
tumors, peripheral neuritis, traumatic neuroses, mania, melancholia, and hyste- 
ria. In this type we find the administration of carbohydrates in the food being 
followed by a glycosuria, because the diastatic processes are continually urged 
to increased activity, to such an extent that the hepatic artery becomes loaded 
with sugar. In other words, we have, in this type, an increased rate of sac- 
charification rather than a diminished formation of glycogen. 1 

In other cases a continuous glycosuria is observed even after complete 
exclusion of carbohydrates from the diet. In this type there is a constant 
formation of carbohydrates from the protein and fat of the food. The liver 
appears to be capable of storing up glycogen in the usual amount, but the 
system in general is practically unable to utilize the sugar brought to it. In 
consequence of this the liver-cells are repeatedly called upon to convert the 
glycogen reserve into glucose, and as a result the blood becomes laden with 
sugar. Even under these circumstances the hyperglycemia would not neces- 
sarily cause a glycosuria, providing the normal ferments of the blood arising 
from the internal secretion of the pancreas were present. This type of glyco- 
suria is in reality typical diabetes mellitus and is so dependent upon such a large 
number of factors that the writer must refer to other works for its discussion. 
Recently Pfliiger has shown that a very close relationship exists between the 
nervous influences of the duodenum and pancreas. His work apparently in- 
dicates that what is generally known as pancreatic diabetes is dependent 
to some extent upon disturbance of the proper correlation between the duo- 
denum and pancreas. A peculiarity in the true diabetic glycosuria is that 
other types of sugar, especially levulose, appear to be well tolerated by the 
diabetic individual without leading to a glycosuria. Besides, the administra- 
tion of the primary hydrolytic products of glucose, as for instance gluconic and 
saccharic acids, apparently diminishes an already existing glycosuria. This 
would seem to indicate that the system is primarily unable to bring about the 
initial cleavage of the glucose molecule. As it has been shown that many 
diabetic cases are associated with sclerosis of the i slands of Lacgerhans in the 
pancreas, this organ is usually regarded as the principal seat of the patho- 
logic changes in true diabetes mellitus. On the other hand, many cases which 
are clinically indistinguishable from those of pancreatic diabetes show no 
lesion in this organ postmortem nor any characteristic lesion in other organs. 2 

Occasionally, cases are seen in which a diplomellituria exists. This is 
the contemporaneous or alternate occurrence of diabetic and non-diabetic 
glycosuria in the same individual (Stern 3 ). The clearing up of one type 
usually reveals the other. A transient glycosuria may obtain in conditions in 
which the oxygen supply is reduced, as in suffocation, poisoning with carbon- 
monoxid, curare and amyl nitrite, or after administration of such drugs as 

1 See Novak, Porges and Strisower, Ztschr. f. klin. Med., 1913, LXXVIII, 413; also, Mann, 
Ibid., 488; Labbe and Bouchage, Lancet, 1914, I, 13. 

2 See Homans, Jour. Med. Research, 1914, XXX, 49; Major, Ibid., 1914, XXXI, 313; 
Bensley, New York Med. Jour., 1915, CI, 523; Kirk, Arch. Int. Med., 1915, XV 39. 

3 Arch. Diagnosis, 1910, III, 236. 



288 DIAGNOSTIC METHODS. 

strychnine, cocaine, caffeine and adrenalin. 1 Further, a transient glycosuria 
is not infrequently seen following the use of alcohol, especially in the form 
of beer or champagne. A post-anesthetic glycosuria is observed after the use 
of chloroform and ether, probably as a result of the action of these agents in 
stimulating the transformation of glycogen into dextrose (Hawk 2 ). 

Qualitative Tests for Glucose. 

The qualitative tests for sugar depend for the most part upon the chemical 
structure of its molecule. The hexoses belong either to the class of aldehyds 
or ketones, and as such will reduce metallic oxids to lower forms. The CHO 
and CO groups of the aldoses and ketoses, respectively, are the reacting points 
in all of the reduction tests, such as those with copper and bismuth solutions, 
as well as in the tests showing the formation of the characteristic osazones. 
Moreover, these carbohydrates show the peculiarity of fermenting, in the 
presence of yeast, into alcohol, carbonic acid, and other products. This 
fermentation test, especially with the saccharomyces cerevisiae, is given 
only by the sugars having three or a multiple of three carbon atoms in the 
molecule. Fischer's work has shown that only those sugars may be fermented 
by a specific ferment in which the ferment and sugar stand to one another 
in such a relation that a chemical union is possible between them or as he 
expresses it, only when the ferment fits into the sugar molecule like a key in 
a lock. This explains why only certain types of the hexoses will ferment in 
the presence of yeast. 

Before any qualitative or quantitative test may be made for the pres- 
ence of sugar in the urine, albumin must be removed, especially if present 
in more than traces. 3 

Trommels Test. 
To a few c.c. of urine in a test-tube are added one-third its volume of a 
10 per cent, solution of sodium hydrate and then, drop by drop, a 10 per cent, 
solution of copper sulphate. This copper sulphate should be added with con- 
stant shaking until a slight excess of the precipitated cupric hydrate (Cu(OH) 2 ) 
remains undissolved and is visible, on shaking the tube, as a distinctly greenish- 
blue flocculent precipitate. The upper layer of the urine is then warmed, 
when a yellow or red precipitate appears in the heated urine if sugar be present. 
As a rule, it is unnecessary and even unwise to boil the solution, as otherwise 
substances other than sugar may produce the reaction. It is true that the re- 
action is not as sensitive unless the solution be heated to boiling, but other sub- 
stances do not so readily interfere with the reaction at a lower temperature. The 

1 See Emerson (Jour. Am. Med. Assn., 1912, LIX, 2245) for a discussion of glycosuria in 
the insane; also, Herbert, Biochem. Ztschr., 1913, XLVIII, 120; Landau, Ztschr. f. klin. 
Med., 1913, LXXIX, 201; Folin, Denis and Smillie, Jour. Biol. Chem., 1914, XVII, 519; 
Luzzatto, Ztschr. f. exp. Path. u. Ther., 1914, XVI, 18; Loewy and Rosenberg, Biochem. 
Ztschr., 1914, LXI, 189; Eustis, Am. Jour. Med. Sc, 1914, CXLVEI, 830; Anders and Jameson, 
Ibid., 1914, CXLVIII, 323; Pozzo, Policlinico, 1915, XXII, 417. 

2 Arch. Int. Med., 1911, VIII, 39. See, also, King, Moyle and Haupt, Jour. Exper. Med., 
1912, XVI, 178; Greenwald, Jour. Biol. Chem., 1913, XVI, 375; Ross and Hawk, Arch. Int. 
Med., 1914, XIV, 779. 

3 See Meinertz, Med. Klin., 1913, IX, 1771; Glaserfeld, Med. Klin., 1914, X, 1388, 1413 
and 1452; Tirard, Lancet, 1914, II, 1133; Geelmuyden, Norsk Mag. f. Laegevidensk., 1915, 
LXXVL 08 s. 



THE URINE. 289 

yellow or red precipitate will gradually form throughout the mixture, from above 
downward, and will finally settle out, leaving a colorless or yellow fluid above. 
This reaction is due to the reduction of the cupric hydrate, which is 
formed by the action of the sodium hydrate upon the copper sulphate, into 
cuprous hydrate, which becomes dehydrated, on heating, into cuprous oxid. 
If no sugar be present in the urine and other reducing substances are not exces- 
sive in amount, the cupric hydrate will settle out on warming as a black pre- 
cipitate of cupric oxid. The equations showing these points are as follows: 

CuS0 4 + 2 NaOH = Na 2 S0 4 + Cu(OH) 2 
Cu(OH) 2 + heat=CuO + H 2 

2CuO + glucose (CHO-(CHOH) 4 -CH 2 OH) = Cu 2 + gluconic 
acid (COOH-(CHOH) 4 -CH 2 OH). 

Whether a yellow or a red precipitate forms will depend upon the alka- 
linity of the solution, the stronger the alkalinity the more pronounced is the 
red color due to cuprous oxid, while in less strong alkaline solutions the yellow 
color of cuprous hydrate will predominate. Certain substances normally 
present in the urine as well as some which may be added to it have the property 
of holding in solution the cupric hydrate first formed. This property is shown 
by the deep blue color which the solution assumes. This color is very intense 
in the presence of sugar, but it is unwise to assume the presence of sugar from 
this fact alone. Among the substances which dissolve cupric hydrate and 
which may be present in the urine in varying amounts we find ammonium 
compounds, albumin, uric acid, creatinin, allantoin, mucin, glucose, lactose, 
maltose, pyrocatechin, hydroquinon, alkapton acids, bile pigments, and gly- 
curonic acid. On warming the solution, which may not contain sufficient 
amounts of glucose to give a typical reaction, a slight reduction of the copper 
solution will occur leading to the formation of a dirty yellow solution. If 
these bodies be present in excess a distinct precipitate may occur, and as a result 
marked confusion may arise regarding the presence of sugar. 1 It has been 
shown that uric acid and creatinin do not readily reduce at as low a temperature 
as does sugar, so that slight warming is much better than boiling. The pres- 
ence of these bodies very frequently leads to a change in color from the bright 
blue to the greenish-yellow which may be due to the presence of sugar in small 
amounts, but should never be regarded as indicative of a pathological glycosuria. 

It should be a working rule, therefore, that mere decolorization of the 
fluid should not be regarded as due to sugar. Glucose reduces so much 
more markedly than the other bodies mentioned that a distinct granular pre- 
cipitate either of cuprous oxid or hydrate forms and settles out, leaving a 
supernatant fluid partially or completely decolorized. Normal urine or 
urine containing an excess of uric acid, creatinin, or ammonium compounds 
practically never produces an immediate precipitate unless the solution be 
boiled for some time. This is due to the fact that these substances hold in 
solution the amount of cuprous oxid which is formed by their reducing action. 

1 See Schulz, Ztschr. f. phvsiol. Chem., 1912, LXXVII, 121; also, Salkowski, Ibid., 1912, 
LXXIX, 164. 

19 



29O DIAGNOSTIC METHODS. 

If such solutions be allowed to stand for a time a reddish-yellow precipitate 
may occur, but this should not confuse as the typical sugar reaction occurs im- 
mediately on warming unless too much copper solution be added. In case 
only traces of sugar be present, we may obtain little or no positive reaction 
because the sugar holds in solution the traces of cuprous oxid formed just as 
do the other substances above mentioned. As a rule, however, sugar in patho- 
logic amounts shows a reducing action over and beyond its dissolving action, 
so that a red or yellow precipitate must settle out. The limit of this test for 
sugar is about 0.2 per cent., in which case the reduction will occur as in normal 
urine without the separation of the characteristic copper precipitate. Even 
in this case the yellow color is somewhat more intense and is clearer than the 
dirty yellow color produced in the presence of excessive uric acid or creatinin. 
If the urine contain an excess of the conjugated glycuronates, the test 
may be quite as distinctive as that for sugar. To differentiate such reactions, 
one should resort either to the phenylhydrazin or the fermentation tests. 

Benedict's Test. 

This qualitative test 1 is, in the writer's opinion, the best modification of 
Trommer's test. The reducing action of glucose in alkaline solution is dimin- 
ished by strong alkalies such as the hydrates. This property may prevent such 
well-known solutions as Fehling's and Haines' (discussed below) from demon- 
strating small quantities of sugar. Further, the urine contains many compounds 
which interfere with the detection of glucose by the strongly alkaline solutions, 
the loss of delicacy in the other tests being probably due to the fact that normal 
reduction of the solution is inhibited for a period long enough to allow the strong 
alkali to decompose the reducing substance. However, in solutions of the 
alkali carbonates the reduction develops more slowly and is not destroyed by 
this weak alkali. The addition of sodium citrate, instead of the Rochelle salt 
as used by Fehling, makes the solution very stable, although little more than 
does the glycerin of Haines' solution. For the reasons given, this solution of 
Benedict is more sensitive (about ten times) to urinary sugar than are the other 
copper solutions. Moreover, it is not appreciably reduced by creatinin and 
uric acid, nor by such preservatives as chloroform, chloral or formaldehyd, 
which introduce errors into the other tests. It is, however, promptly reduced 
by the alkapton acids and the conjugated glycuronic acids. The formula of 
the reagent is as follows: 

Copper sulphate (C. P. crystallized), 17.3 grams 
Sodium or potassium citrate, !73-° grams 

Sodium carbonate (crystallized), 2 200.0 grams 
Distilled water, q. s. ad., 1000.0 c.c. 

Dissolve the citrate and carbonate (with aid of heat) in about 700 c.c. of water 
and filter if necessary. Dissolve the CuS0 4 in about 100 c.c. of water and pour 
into the alkaline solution. Cool and make up to 1 liter. 

1 Jour. Am. Med. Assn., 1911, LVII, 1193. See, also, Kleiner, Ibid., 1914, LXII, 1307; 
Sheaff, Ibid., 1915, LXV, 1181; Weinberger, Am. Jour. Med. Sc, 1914, CXIATI, 407. 

2 Or one-half the amount of the anhydrous salt may be used. 



THE URINE. 29I 

The technic is as follows: 5 c.c. of the reagent are placed in a test-tube and 
not more than 8 or 10 drops of urine are added. The mixture is then heated to 
vigorous boiling for one to two minutes and allowed to cool spontaneously. In 
the presence of glucose, the entire body of the solution will be filled with a 
precipitate which may be red, yellow or greenish in tinge. If the quantity of 
glucose be low (under 0.3 per cent.) the precipitate forms only on cooling. If 
no sugar is present, the solution either remains perfectly clear or shows a faint 
turbidity, which is blue in color and consists of precipitated urates. This test 
may be especially recommended for all qualitative work. 

Fehling's Test. 

This is, perhaps, the best-known test for sugar but it is inconvenient and 
frequently inaccurate for the reasons above mentioned. The formulae for the 
solutions are as follows: 

Solution A. Solution B. 

Copper sulphate, 34.64 gm. Rochelle salt, 173 gm. 

Distilled water, q. s., ad., 500 c.c. Sodium hydrate 50 gm. 

Distilled water, q. s., ad., 500 c.c. 

In performing the test with Fehling's solution equal parts of solutions A 
and B are taken and the mixture brought to a boil. The urine is then added, 
drop by drop, when a reduction of the copper solution will appear in the pres- 
ence of sugar. 1 The amount of urine added should rarely exceed 10 drops, 
at the outside 20, if a reduction is to be taken as typical and the solution should 
not be boiled for more than a few seconds after adding the urine. The addi- 
tion of larger volumes of urine will usually introduce errors from the factors 
mentioned under Trommer's test. This test has the same points of inter- 
est as has Trommer's test, so that it is wise to dilute the urine before making 
the test. It is more frequently used than is Trommer's test, but it is not as 
convenient. 

Haines' Test. 

Haines has introduced a modification of Trommer's test by adding glycerin, 
instead of Rochelle salt, to increase the amount of copper in solution. This 
test is much more convenient than Fehling's, the solution having the advantage 
of keeping almost indefinitely. It is, however, far less delicate than is Benedict's 
and is reduced by preservatives as well as by excess of many normal urinary 
constituents, especially by uric acid and creatinin. 2 

The composition of Haines' qualitative solution is as follows: 

Copper sulphate, 12 grams 

Potassium hydrate, 3 45 grams 
Glycerin, 90 c.c. 

Water, q.s., ad., 1000 c.c. 

1 See Cramer, Biochem. Jour., 191 5, IX, 71. 

2 The creatinin may be almost entirely removed from the urine, if desired, by adding to 10 
c.c. of urine about 2 grams of picric acid and about 2 grams of Merck's blood charcoal, shaking 
for a few minutes and filtering (see Folin, Jour. Biol. Chem., 1915, XXII, 327). 

3 Or 32 grams of sodium hydrate. 



292 DIAGNOSTIC METHODS. 

A perfectly clear, transparent, dark-blue liquid results which throws 
down a very slight reddish deposit of cuprous oxid on standing a week or 
more. This does not affect the value of the solution, as the clear blue solu- 
tion is simply decanted as required. 

Four or five c.c. of this solution are placed in a test-tube and gently boiled. 
Six drops of the suspected urine are added and the upper portion of the mix- 
ture brought to a boil and immediately removed from the flame. If sugar be 
present an abundant yellow or yellowish-red precipitate is thrown down; 
if no such precipitate occurs sugar is absent. The precautions to be observed 
in using this test are never to add at the outside more than 10 drops of urine 
and not to boil the mixture for more than one or two seconds after the addition 
of urine. 

Many other modifications of the copper test have been introduced, both 
for qualitative and quantitative purposes, but they do not have any advantages 
over those mentioned especially over Benedict's test. This latter is used daily 
in the writer's laboratory and has been found very reliable and serviceable as 
it does not react with the usual preservatives which are added to urine. When 
the other tests are used an error may creep into the report which can be corrected 
only by resort to fermentation and other confirmatory methods. 
Almen-Nylander's Test. 

This test is a modification of the original Bottger test and is a distinct 
improvement. The reagent is prepared as follows: Four grams of Rochelle 
salt are dissolved in 100 c.c. of 10 per cent, sodium hydrate solution with gentle 
heat, and as much bismuth subnitrate is added as will dissolve (about 2 grams). 
After the mixture is cooled the undissolved bismuth subnitrate is filtered off 
and the filtrate kept in a dark bottle, where it will remain permanent for a 
long period. 

To a few c.c. of urine in a test-tube is added one-tenth of the volume of 
this reagent and the mixture boiled for a few minutes. If glucose is present, 
the fluid will darken and a black precipitate of metallic bismuth separate 
out. This black precipitate must occur while the solution is being warmed and 
not after it has cooled. If only a small amount of sugar be present the phos- 
phates precipitated by the alkali may be slightly gray in color instead of the 
usual white. This test is somewhat more delicate than is the copper test, as 
it will show about 1/40 per cent, of sugar. 

This reagent is not reduced by uric acid, creatinin, pyrocatechin, hydro- 
quinon or homogentisic acid; but it is reduced by the conjugated glycuronic 
acid, excess of urinary pigment and pentoses. It is particularly necessary in 
this test that albumin be removed, as bismuth sulphid forms in the pres- 
ence of albumin, and may precipitate either in the form of a reddish or a dis- 
tinctly brownish, even black precipitate. The reduction observed after the 
administration of medicaments, such as rhubarb, senna, antipyrin, camphor, 
salicylic acid, salol, sulphonal, trional, quinin, eucalyptus, oil of turpentine, 
and chloral hydrate, is usually a brown rather than a black unless the solution 
stands for some time. The administration of saccharin usually results in a 
reduction of this test, while the copper tests are not affected by it. Sulphur 



THE URINE. 293 

bodies (methyl mercaptan) excreted after the patient has eaten asparagus give 
a distinct precipitate which may be more or less confusing. If the urine be 
ammoniacal the reaction may not appear in a characteristic way owing to the 
fact that the free ammonia is evolved and the alkalinity of the solution is reduced 
by the combination of the sodium ion with the acid radical formerly bound 
to the ammonium ion. 

This test is used by many as a routine test for sugar, as it is practically 
always negative with normal urine. The reducing action of the glycuronates 
and pentoses must, however, be remembered. 1 

Fermentation Test. 

As previously stated, the reduction tests do not absolutely prove the pres- 
ence of sugar. All that one can say is that a reducing substance is present and 
if the reaction be typical the probability is that the reducing substance is sugar. 
The fermentation test is, perhaps, the most certain of all the tests for glucose 
and depends upon the fact that only those sugars which contain three or a 
multiple of three carbon atoms are fermentable with yeast. 2 Not all members 
of these groups of sugars will ferment with yeast, so that for absolutely scientific 
purposes fermentation will not differentiate them. Fischer's work along this 
line should be carefully read by anyone interested in the biologic properties 
of the various types of sugar. As the hexoses, which occur in the urine are 
practically limited to glucose and levulose, we are safe in saying that any 
sugar fermenting with yeast is one or the other of these monosaccharides, which 
may be differentiated by tests to be outlined later. 

The test is performed as follows: Ten c.c. of urine are placed in a test- 
tube and a piece of compressed yeast, which should be perfectly fresh, about 
the size of a pea is added and the urine gently shaken until the yeast is finely 
divided. This mixture is then poured into a fermentation tube, which is 
allowed to stand in the incubator for a few hours. Two control tests, using 
in one normal urine and yeast and in the other normal urine, yeast, and a 
trace of dextrose, are then made and placed in the incubator along with the 
suspected urine. The presence of sugar is indicated by gas (C0 2 ) in the upper 
portion of the fermentation tube. The rapidity of formation of this gas depends 
upon the amount of yeast as well as upon the age of the yeast. The test in- 
dicates from 0.1 to 0.05 per cent, of sugar, especially if the urine be sterilized 
by previous boiling. The compressed yeast as usually purchased develops 
a certain minimal amount of gas in normal urine, so that the control test is 
necessary both to show whether " self -fermentation" is excessive and also 
whether the yeast is at all active in producing C0 2 from glucose when it has 
been added. It is necessary, moreover, that decomposition of the urine be 
prevented, either by previously boiling the urine, addition of a trace of sodium 
fluorid, or tartaric acid. 

This test has the great advantage that the other substances, which reduce 
copper and bismuth solutions, do not ferment. If precautions are observed 

1 See Mende, Munch. Med. Wchnschr., 1914, LXI, 1120. 

2 Boros (Med. Klin., 1913, IX, 874) shows that yeast cells may be so numerous in the urine, 
as voided, to destroy the sugar present. This is known as masked or occult glycosuria. 



294 DIAGNOSTIC METHODS. 

to add the right amount of yeast, not to shake the yeast and urine violently 
enough to include much air, and to prevent bacterial decomposition, this test 
will positively show the presence or absence of glucose or levulose in the urine 
and no other substances. It is always wise to use this test either as confirma- 
tory or decisive in conjunction with the previous reduction tests. 

Phenylhydrazin Test. 

This test is much more delicate than any of the previous tests mentioned. 
Theoretically it will show sugar in the amount present in normal urine, but 
practically no definite reaction is observed. The principle of the reaction is 
the decomposition which occurs between the aldehyd or ketone group of the 
sugar molecule and the amino group of the phenylhydrazin. In this reac- 
tion, characteristic bodies known as hydrazones are first formed, which are 
converted, in the presence of dilute acetic acid and an excess of phenylhydra- 
zin, into crystalline bodies known as osazones. These latter bodies are char- 
acteristic of the sugar group, usually differing from one another, depending 
upon the original sugar from which they were formed. The osazones crystal- 
lize in definite forms, the purified crystals showing rather sharp melting points. 
It is, therefore, essential not only that a crystalline body be obtained in this 
reaction, but that the crystalline form and the melting-point of the crystal be that 
characteristic of the sugar suspected. The success of the test will depend 
largely on the relation of the sugar to the reagents, the best proportions being 
approximately one of sugar, two of phenylhydrazin, and three of sodium 
acetate. All of the members of the hexose and pentose groups show this 
reaction, as do many of those of the polysaccharide series. As will be seen from 
the reaction given below, those sugars which differ only in the space-relations 
of the atoms attached to the first two carbon atoms can possibly give the same 
osazone. For this reason we find glucose, levulose, and mannose as well as 
glucosamine giving exactly the same osazone (phenylglucosazone), showing 
a characteristic yellow needle-shaped crystalline deposit, with a melting-point 
of 204 to 205 C. The reactions leading to the formation of this body are 
shown in the following equations: 

CH 2 OH-(CHOH) 4 - CHO + NH, - NHC 6 H 5 = CH 2 OH-(CHOH) 4 - 
CH = N - NHC 6 H S + H.0 
CH 2 OH - (CHOH) 3 -CHOH - CH = N - NHC 6 H 5 + NH 2 - NHC 6 H 5 =- 
CH 2 OH-(CHOH) 3 -C-CH + 2H 2 

II II 
C 6 H 5 NH— N N— NHC 6 H S 

Twenty-five c.c. of urine are treated with a few drops of a solution of lead 
acetate and filtered to remove the albumin if present. To the filtrate, which must 
be acidified with acetic acid if it is not already acid, is then added phenyl- 
hydrazin hydrochlorate (1/2 to 1 gram) and about 2 grams of sodium acetate. 
The tube is then shaken thoroughly to mix the contents and is placed in a 
boiling water-bath for from one to two hours. Some workers recommend a 
shorter period, such as 20 minutes, but the writer has never been able to get 
as good results with the short heating. At the end of this time the material in 



PLATE IX. 




Osazons. {Hawk.) 
Upper form, dextrosazon; central form maltosazon; lower form, lactosazon. 



THE URINE. 295 

the tube is filtered while hot and the filtrate allowed to cool slowly rather than 
as some advise to cool suddenly by immersion in cold water. If glucose be 
present a yellow crystalline deposit will appear, which under the microscope 
will show the characteristic needle-shaped crystals arranged in bundles or 
sheaves. This microscopic examination does not absolutely prove the crys- 
tals to be phenylglucosazone so that a melting-point determination must be 
made before definite proof is forthcoming. For this purpose the crystals must 
be purified by dissolving in a hot 60 per cent, alcohol and recrystallizing by 
adding water and evaporating the alcohol. A few of these crystals are then 
placed in a perfectly dry capillary tube and the melting-point determined by 
methods previously learned in organic chemistry. 

Neumann has applied Fischer's method of using this test to the urine. 
Five c.c. of urine are treated in a test-tube with 2 c.c. of a 50 per cent, solution 
of acetic acid saturated with sodium acetate and two drops of pure phenyl- 
hydrazin. This mixture is then evaporated by boiling to 3 c.c, after which 
it is cooled quickly and is then rewarmed and allowed to cool slowly. If 
glucose be present to the amount of 0.02 per cent., pure crystals of phenyl- 
glucosazone separate out in 5 to 10 minutes. If the urine has a high specific 
gravity and a low sugar content the crystals do not form so quickly. This 
test is much to be preferred in well-equipped laboratories, but is not as conven- 
ient as the use of the crystalline phenylhydrazin hydrochlorate for the general 
worker, as the fluid phenylhydrazin is quite irritating and is not as. easy 
to work with. 

This test cannot be used for quantitative purposes as the yield is never 
complete. For qualitative purposes, however, it is to be especially recom- 
mended. When properly applied it is the most delicate and one of the most 
reliable tests at our disposal. If the melting-point of the crystals be determined 
the only confusing substances will be levulose, glucosamin, and mannose. 
The osazones formed from the other carbohydrates and the glycuronic acid 
compounds crystallize in somewhat similar form, but do not show the character- 
istic melting-point of 204 to 205 C. of the phenylglucosazone. It is true 
that the impure crystals melt at somewhat lower temperature, but if carefully 
purified from 60 per cent, alcohol they will melt at approximately 204 . 

Quantitative Methods. 
Fehling's Method. 

The solutions to be used in this test have been given under the qualitative 
test for sugar. The principle of the test is the determination of the exact 
amount of sugar necessary to decolorize a mixture of 5 c.c. each of solutions 
A and B. Providing chemically pure copper sulphate has been used in pre- 
paring solution A, the mixture is reduced by an amount of urine which con- 
tains 0.05 gram of glucose. If there is any question regarding the purity of 
the copper sulphate, the strength of the solution must be determined by titrat- 
ing against a known solution of chemically pure glucose, or of cane-sugar, 
which has been inverted by heating with dilute acid (see Sutton's Volumetric 
Analysis) . 



296 DIAGNOSTIC METHODS. 

Technic. 

Five c.c. each of solutions A and B (p. 291) are carefully measured from 
a buret or pipet into an Erlenmeyer flask of about 250 c.c. capacity. Forty 
c.c. of distilled water are then added and the mixture boiled. The urine is 
now added in small quantities from a buret to this boiling copper solution 
until the blue color has entirely disappeared. It is advisable to add the urine 
rather rapidly as a preliminary test, it being practically impossible with a single 
titration to obtain accurate results. In doing this one adds a c.c. of urine 
at a time and observes whether the color still remains. If so, the titration 
is continued, adding from 1/2 to 1 c.c. at each period, until the color has dis- 
appeared. The number of c.c. used is then observed and a second test made 
as follows: To the boiling copper solution is added directly 1 c.c. less of 
urine than was used for complete decolorization in the preliminary test. The 
urine is then added two drops at a time, noting after each addition whether 
the color remains. In this way an accurate end-point may be reached. 

As the amount of urine used to decolorize the 10 c.c. of copper solution is 
equivalent to 0.05 gram of glucose, a simple calculation yields the percentage 
and total values for glucose in the urine. As this test is not accurate with high 
concentrations of the urine, it is necessary that the sugar content should range 
between 1/2 and 1 per cent. Dilution may, therefore, be necessary. A good 
working rule is to dilute a urine of a specific gravity of 1030 five times (by 
adding 4 volumes of water) and ten times if the specific gravity be above 1035. 
There is little danger of getting the dilution too great to show the characteristic 
reaction for sugar, as this test shows the presence of sugar when present to the 
amount of 0.08 per cent. In the use of this method, as well as of most reduction 
tests, albumin must first be removed if present in more than traces. Moreover, 
the presence of preservatives as well as excess of uric acid or creatinin will increase 
the reducing power of the urine as determined by this test, a considerable error 
being occasionally introduced unless overcome by dilution or removal of the 
interfering substances. 

Bang's Method. 

This method 1 appears to the writer to be the best one at our disposal for 
the quantitative estimation of sugar in the urine. Like the solution of Benedict 
previously mentioned, it contains alkali carbonates instead of hydrates. The 
principle of this method is as follows: The urine is treated with an excess of 
standard alkaline copper solution and boiled to bring out the reduction due 
to sugar. The amount of copper remaining in excess is then determined by 
titration with a solution of hydroxylamin sulphate (instead of with potassium 
sulphocyanate as used by Citron) and from this the amount of sugar is 
calculated. 

The solutions necessary in this test are as follows: 

(a). One hundred grams of potassium bicarbonate are dissolved in about 
1300 c.c. of distilled water contained in a 2-liter flask. To this solution are 

1 Biochem. Ztschr., 1907, II, 271; 1908, XI, 538; 1911, XXXII, 443. Bang (Ibid., 1913, 
XLIX, 1) has devised a new method which is cheaper and equally exact. 



THE URINE. 297 

added 500 grams of potassium carbonate and 400 grams of potassium sulpho- 
cyanate. Exactly 25 grams of pure copper sulphate (CuS0 4 5H 2 0) are then 
dissolved in about 150 c.c. of warm distilled water. After cooling this solution 
is added quantitatively to the carbonate solution. Add water up to the 2-liter 

BANG'S TABLE OF REDUCTION EQUIVALENTS. 



Cubic centimeters of 


Milligrams of 


Cubic centimeters of 


Milligrams of 


hydroxylamin solution 


sugar 


hydroxylamin solution 


sugar 


o-75 


60.0 


25-5o 


23-5 


1 .00 


59-4 


26.00 


22.9 


1.50 


58.4 


26.50 


22.3 


2.00 


57-3 


27 .00 


21.8 


2.50 


56.2 


27.50 


21 .2 


3.00 


55-o 


28.00 


20.7 


3-5° 


54-3 


28.50 


20.1 


4.00 


53-4 


29.00 


19.6 


4-5° 


52.6 


29.50 


19. 1 


5.00 


5i.6 


30.00 


18.6 


5-5° 


50.7 


30-50 


18.0 


6.00 


49-8 


31.00 


17-5 


6.50 


48.9 


3i-5o 


17.0 


7 .00 


48.0 


32.00 


16.5 


7-5° 


47.2 


32.50 


15-9 


8.00 


46.3 


33- 00 


15-4 


8.50 


45-5 


33-5o 


14.9 


9.00 


44-7 


34.00 


14.4 


9-5° 


44 .0 


34-5o 


^3-9 


10.00 


43-3 


35 -oo 


13-4 


10.50 


42.5 


35-5o 


12.9 


11 .00 


41.8 


36.00 


12.4 


11.50 


41. 1 


36.50 


11. 9 


12.00 


40.4 


37.00 


11. 4 


12.50 


39-7 


37-5o 


10.9 


13.00 


39-° 


38.00 


10.4 


13.50 


38.3 


38.50 


9.9 


14.00 


37-7 


39.00 


9.4 


14.50 


37-i 


39-5o 


9.0 


15.00 


36.4 


40.00 


8-5 


i5-5o 


35-8 


40.50 


8.1 


16.00 


35-i 


41 .00 


7.6 


16.50 


34-5 


41.50 


7-2 


17 .00 


33-9 


42.00 


6.7 


i7-5o 


33-3 


42.50 


6-3 


18.00 


32.6 


43.00 


5-8 


18.50 


32.0 


43-5° 


5-4 


19.00 


3i-4 


44.00 


4-9 


19.50 


30.8 


44-5o 


4-5 


20.00 


30.2 


45.00 


4.1 


20.50 


29.6 


45-5o 


3-7 


21 .00 


29.0 


46.00 


3-3 


21.50 


28.3 


46.50 


2.9 


22.00 


27.7 


47- 00 


2-5 


22.50 


27 .1 


47-5o 


2.1 


23.00 


26.5 


48.00 


i-7 


23-50 


25-8 


48.50 


i-3 


24.00 


25.2 


49.00 


0.9 


24.50 


24.6 


49-5o 


0.5 


25.00 


24.1 


50.00 


0.0 



For every 1/10 c.c. hydroxylamin solution used more than is given in the table, subtract 
0.1 mg. if the reading be between 49 and 15, while if it be between 15 and 1 substract 0.2 mg. 



290 DIAGNOSTIC METHODS. 

mark, allow to stand for 24 hours and filter. This solution is stable for about 
three months. 1 

(b). Two hundred grams of potassium sulphocyanate are dissolved in 
about 1500 c.c. of distilled water in a 2-liter flask. 6.55 grams of hydroxylamin 
sulphate are dissolved in water and added quantitatively to the sulphocyanate 
solution. Add water up to the 2-liter mark and preserve the mixture in dark 
colored bottles. This solution is very permanent. 

Technic. 

Ten c.c. of urine (5 or 2 c.c. diluted with water to 10 c.c. if more than 0.6 
per cent, sugar is present) are measured into a 200 c.c. Jena Erlenmeyer flask 
and treated with 50 c.c. of the copper solution (a). Heat on a wire gauze to 
boiling for exactly three minutes. Cool quickly to room temperature by immer- 
sion of the flask in cold water. Now titrate with the hydroxylamin solution (b) 
until a colorless mixture results. From the number of c.c. of hydroxylamin sul- 
phate solution used, calculate the sugar in milligrams by means of the accom- 
panying table. A simple calculation yields the percentage and total amounts. 

It has been found difficult to get a sharp end-point in this reaction if the 
urine be rather highly colored. Bang and Bohmansson 2 have shown that the v 
urine may be cleared by the following process. Twenty c.c. of urine are treated 
with 5 c.c. of 25 per cent. HC1 and 2 grams of blood charcoal. Shake a few 
times during five minutes and filter through a dry filter into a dry beaker. This 
filtrate is then used for the test as described above. Woodyatt and Helmholz 3 
and Andersen 4 have called attention to the necessity of testing the charcoal by 
control experiments before using the above method of clearing. As some of the 
blood charcoals give very erroneous results, they recommend the use of Merck's 
preparation. A further advantage of using this clearing method is that the 
"auto-reduction" of the urine is reduced to a low limit owing to the almost 
complete removal of urochrom, uric acid, creatinin and glycuronic acid 
derivatives. 5 

Purdy's Method. 

It has been found that the addition of strong ammonia to the mixed 
copper and tartrate solutions makes the end-point much more distinct. Pavy, 
Sahli, Kumagawa and Suto, Kinoshita, and others have modified the original 
Fehling's solution in this way. It was shown, however, by Lowe that the 
substitution of glycerin for the Rochelle salt made a much more stable solution 
and one which could be originally mixed and kept for indefinite lengths of 
time. Purdy has succeeded in obtaining a copper solution which, in the writer's 
experience, is much to be preferred for general laboratory purposes over most 
of the other modifications of Fehling's solution. His formula is as follows: 

1 Hatta (Biochem. Ztschr., 1913, LII, 1) show's that the self-reduction of this solution may 
give as high an error as 9% on the positive side. 

2 Ztschr. f. physiol. Chem., 1909, LXIII, 443; Biochem. Ztschr., 1909, XIX, 281. 

3 Arch. Int. Med., 1911, VII, 598. 

4 Biochem. Ztschr., 1911, XXXVII, 262. 

5 Bang (Biochem. Ztschr., 1912, XXXVIII, 168) has recently advised the substitution of 
2 c.c. of 95 % alcohol for the 5 c.c. of HC1. 



THE URINE. 299 

Chemically pure copper sulphate, . 4.752 grams 

Potassium hydrate, 1 23.500 grams 

Strong C. P. ammonia (sp. g. 0.88), 350.000 c.c. 

Glycerin, 38.000 c.c. 

Distilled water, q. s. ad., 1,000.000 c.c. 

This solution is prepared by dissolving the copper sulphate and glycerin 
in 200 c.c. of distilled water, heating gently if necessary. The potassium 
hydrate is dissolved in a second 200 c.c. of water and mixed with the copper 
solution. When the mixture has cooled add the ammonia and bring the total 
volume up to 1 liter with distilled water. Thirty-five c.c. of this solution are 
decolorized by 0.02 gram of glucose. 

Haines has slightly modified this original solution of Purdy so that 10 c.c. 
of the solution are decolorized by 0.01 gram of glucose. This titer is somewhat 
more convenient than that of Purdy as the calculation is distinctly simplified. 
The formula for his modification is as follows: 

Pure copper sulphate, 8.314 grams 

Pure potassium hydrate, 2 25.000 grams 

Glycerin, 40.000 c.c. 

Ammonia, 350.000 c.c. 

Distilled water, q. s. ad., 1,000.000 c.c. 

The principle of this test depends upon the fact that, in the reduction of 
cupric oxid in solutions of definite strength by glucose, the blue coloration 
disappears on the addition of a definite amount of glucose without any attendant 
precipitate, the reduced solution remaining transparent and colorless. 

Technic. 

Thirty-five c.c. of Purdy 's test solution or 10 c.c. of Haines' solution 
are measured into an Erlenmeyer flask and 50 c.c. of water added. The 
flask is then closed with a doubly perforated rubber stopper, through one hole 
of which passes the stem of a buret containing the suspected urine and 
through the other a bent glass tube to conduct the fumes of ammonia away 
from the observer. The object of closing the flask with the stopper is to 
exclude the air and thus prevent reoxidation of the cuprous oxid. The contents 
of the flask are now brought to a gentle boil and the urine added, 1 c.c. at 
a time, shaking the flask after each addition until the fluid is completely decolor- 
ized. The number of c.c. used is then noted and a second test made, add- 
ing at once 1 c.c. less of urine than the total number of c.c. used in the 
first experiment. The last portions of the urine are added two drops at a 
time, allowing from three to five seconds to elapse between the addition of these 
separate portions. When the urine is completely decolorized the number of 
c.c. used is noted. As the amount of urine used is equivalent to 0.02 gram 
of glucose with Purdy's solution or 0.01 gram with Haines' solution, the per- 
centage may be obtained as in the previous method. It is necessary with this 
method that the urine be diluted for the same reasons as previously mentioned. 

1 Or 16.8 grams of sodium hydrate. 

2 Or 18 grams of sodium hydrate. 



3°° 



DIAGNOSTIC METHODS. 



In all of the copper tests for glucose the influence of preservative agents 
must be remembered. Thus chloroform, chloral hydrate, and formalin 
will all reduce copper solutions so that an error may be introduced unless these 
substances are removed. 

In selecting a method for the quantitative determination of sugar in the 
urine, the general worker should remember that the method used should be 
both simple and exact. With these points in view, the writer has little hesitancy 
in recommending Bang's method. If the urine be cleared as previously de- 
scribed, the results are all that could be desired, the error being extremely 
small. Many other modifications of the copper tests have been advocated, but 
the writer does not feel that they have any advantage from either the scientific 
or clinical standpoint. The method of Allihn, in which a copper solution is 
reduced and the precipitated cuprous oxid either weighed as such or further 
reduced to metallic copper in a stream of hydrogen, is not clinically available. 
The modifications of Rudisch, Rudisch and Celler, and of Gerrard and Allan 
do not give as accurate results as does the Bang method in the experience of the 
writer. The recent method of Benedict 1 is apparently quite reliable. 
Polariscopic Method. 

In this test the urine must be absolutely clear and must contain no albumin. 
Moreover, it is essential that such substances as glycuronic acid and /9-oxy- 
butyric acid be removed before polarization as they will introduce considerable 




Fig. 84. — One form of Laurent Polariscope. {Hawk.) 

B, Microscope for reading the scale; C, a vernier; E, position of the analyzing Nicol 

prism; H, polarizing Nicol prism in the tube below this point. 

error in the reading. The best clearing agent is that, previously mentioned 
under Bang's test, of shaking the urine with HC1 and blood charcoal and filter- 
ing. Instead of this method the urine, acidified with acetic acid, may be treated 
with a solution of normal lead acetate, which will precipitate the albumin and 
remove excess of pigment. The mixture is filtered and the clear filtrate is used 
for the test. It must be remembered that a correction must be made in the 
percentage of sugar in the filtrate, if the urine be cleared with a solution of lead 
acetate. This may be done by adding 25 c.c. of a 10 per cent, solution of lead 
acetate to 75 c.c. of urine. The sugar in the filtrate will represent only three- 
1 Jour. Biol. Chem., 1911, IX ; 57; Jour. Am. Med. Assn., 1911, LVII, 1193. 



THE URINE. 3OI 

fourth of that of the original urine. Basic lead acetate should be used with 
caution, as it precipitates various sugars. If the urine contain no albumin, 
magnesium oxid or silicic acid may be used as clearing agents. 

Various types of polarimeters have been suggested and most of them 
are quite satisfactory for the determination of sugar. One of these with 
its description is seen in the accompanying cut and legend. The principle 
upon which their use depends is the fact that optically active substances when 
in solution have the power of turning the plane of polarized light either to the 
right or the left. The zero point of the instrument is determined by observing 
the point at which the halves of the optical field have exactly the same degree 
of illumination, when the light passes through a tube either empty or con- 
taining an optically negative fluid. The point at which the graduated scale 
of the instrument and the vernier correspond is regarded as the zero-point. 
In the use of the polariscope any deviation of the plane of light passing through 
the polarizing Nicol prism will be noticed by a darkening of one portion of 
the field, so that the compensating or analyzing Nicol must be rotated until 
both parts of the field are equally illuminated. In. this way one readily deter- 
mines how much the plane of polarized light has been deviated, by observing 




EO 



Fig. 85. — Diagrammatic representation of the course of light through the Laurent 
polariscope. (Direction reversed from that of previous figure.) (Hawk.) 

a, Bichromate plate to purify the light; b, the polarizing Nicol prism; c, a thin quartz 
plate covering one-half the field and essential in producing a second polarized plane; d, tube 
to contain the liquid under examination; e, the analyzing Nicol prism; / and g, ocular lenses. 

the degree of rotation necessary to bring the two portions of the field into equal 
illumination when the light passes through an optically active fluid. The 
most reliable instruments for general work are known as the "half-shadow" 
types, but they rarely find a place in the equipment of the general worker. 
The examination is usually made in a dark room, the light passing through 
the tube ot the polariscope from a sodium flame. 

It must be remembered that the urine may contain substances other than 
glucose which rotate the plane of polarized light. The normal urine is slightly 
levorotatory, the degree varying between 0.05 and 0.18. Albumin is also 
levorotatory and will interfere markedly with the degree of rotation refer- 
able to glucose, unless it be very slight in amount or be entirely removed. 
Levulose, /3-oxybutyric acid, and the conjugated glycuronates are also levo- 
rotatory, the second being especially prone to interfere with the glucose rotation 
as it so frequently is associated with glucose in diabetic conditions. If the 
urine has been previously heated with acid before being examined in the polari- 
scope, glycuronic acid will also interfere, as it shows distinct dextrorotation. 
Cane-sugar as well as lactose are occasionally found in the urine and may 
lead to confusing results as both are dextrorotatory substances. It is, there- 
fore, necessary before any reliable results may be obtained with the polari- 



3° 2 



DIAGNOSTIC METHODS. 



scope to remove the interfering substances, either as preliminary to the de- 
termination of glucose or subsequently, a correction being then made for the rota- 
tion of the interfering substance. It is to be said that the polariscope is more 
fitted for a clinical laboratory than for the general practitioner. 

The rotation of light when passing through optically active solutions is 
dependent upon several factors, among which we find the temperature at which 
the observation is made, the length of tube through which the light passes and 
the concentration of the active solution. By specific rotation of a fluid is 
meant the rotation observed when light passes through a solution containing 
i gram of the active substance per c.c. of fluid and placed in a tube one dcm. 
in length. Thus we find the specific rotation of glucose in such a tube being 
(°)d = +5 2 -74- It i s > therefore, evident that the percentage of sugar in an 
unknown solution contained in a tube i dcm. in length may be obtained by 
dividing the degree of rotation by 0.5274. Some of the tubes for clinical pur- 
pose are constructed of such a length (188.6 mm.) that one degree of rotation 
equals 1 per cent, of glucose. For a full discussion of the subject of optical 
activity of fluids the writer would refer to the work of Landolt. 1 
Technic. 
Having determined the zero-point of the instrument, the tube, which should 
be thoroughly cleaned and dry, is filled with the fluid to be examined. In 
filling this tube precautions must be taken not to include any bubbles of air, 
which is best done by filling the tube until a convex 
iv- 1 meniscus is observed and then sliding the glass disk over 

the end in such a way that the excess of fluid is shoved 
off. The metal cap is then screwed on tightly, but in 
such a way that undue compression is not exerted upon 
the glass disk. The tube is placed in position, the field 
distinctly focussed and the degree of rotation deter- 
mined by revolving the analyzing Nicol until the two 
portions of the optical field have exactly the same 
intensity of illumination. This will require consider- 
able experience, as the accuracy of the determination 
will depend not only upon the clearness of the fluid, the 
degree of focussing, the sensitiveness of the instrument, 
and the brightness of the light, but also upon the sen- 
sitiveness of the observer's eyes. Several readings 
should be made, turning the prisms from both direc- 
tions and observing the degree at which the fields are 
isochromatic, the average being taken as the final result. 
As the eyes soon become fatigued, they should be used 
for only a few seconds at a time. 
Having determined the degree of rotation, the percentage of sugar is then 
calculated as previously mentioned. This method, if carefully used and inter- 
fering substances avoided, is the most accurate method for the determination cf 
glucose. It is, however, difficult, requires much experience, and is not as sensi- 
1 Das optische Drehungs-Vermogen, Braunschweig, 1898. 




Fig. 86. — Einhorn sac- 
charometer. 



THE URINE. 303 

tive as the other methods, rarely detecting the presence of less than 0.2 per 
cent, of glucose. Theoretically, the percentage of sugar as determined by the 
polariscope should exactly agree with that obtained by titration of copper solu- 
tions, but in general work such is never the case, as interfering substances may 
be present or have not been completely removed. 

The writer must refer elsewhere for methods of correcting the readings of 
the polariscope when interfering substances are present, as he does not think 
it wise to recommend the general worker to waste his time with this instrument 
when sufficiently accurate clinical results may be obtained by methods which are 
for him much easier and less liable to error. 

Fermentation Methods. 

The principle of this method has been previously outlined. It has been 
found that, if the precautions mentioned are observed, carbon dioxid is evolved 
quantitatively from glucose by the action of yeast. The most convenient 
method of applying the test is to use an Einhorn fermentation tube which is so 
graduated that the amount of C0 2 evolved is directly read off in terms of per 
cent, of glucose. For this purpose the urine must contain less than 1 per cent, 
of glucose, the urine being diluted as previously described to bring the amount 
within this figure, multiplying the result, of course, by the degree of dilution. 
The urine, which should be acid in reaction, is shaken with a piece of com- 
pressed yeast about the size of a small pea, all precautions being observed as 
mentioned above. The mixture is then poured into the fermentation tube in 
such a way that no air-bubbles collect at the upper end of the tube. Controls 
as previously described are then made and the three tubes placed in the incu- 
bator at 37 C. In a few hours bubbles of gas (C0 2 ) will collect at the top jof 
the tube, the fermentation being practically complete overnight. The percent- 
age of sugar is then read direct from the calibration (1/4-1 per cent.) on the 
tube (the figures from one to five representing c.c. of gas and not per cent, of sugar) . 

Lohnstein's Saccharometer. 

This apparatus is seen in the accompanying cut. Twelve c.c. of mercury 
are placed in the bulb of the apparatus. One-half c.c. of the urine to be 
tested is then floated upon the mercury and treated with a thick paste of com- 
pressed yeast diluted two or three times with water. The stopper is then 
carefully greased with vaseline and inserted so that the two apertures corre- 
spond. By tipping the apparatus a trifle the column of mercury in the long 
tube is then adjusted to the zero point of the scale. When this is done the stop- 
per is turned so that the holes no longer correspond and the weight is placed on 
the stopper to prevent leakage from the increased pressure of the gas liberated 
in the fermentation. The apparatus is then placed in the incubator and the 
extent of fermentation read off by noticing the height to which the column of 
mercury rises in the long arm of the instrument. The fermentation is usually 
complete within six hours. After removing the apparatus from the incubator 
it is allowed to stand in the air for a few minutes to adjust itself to room tem- 
perature, as the scale is graduated in this way. 

This method gives results which correspond very closely to those of titra- 



3°4 



DIAGNOSTIC METHODS. 



tion and is to be recommended for the quantitative determination by fermenta- 
tion methods. 

Lohnstein has also introduced a saccharometer which may be used with 
diluted urine. It is seen in the accompanying cut. It does not, in the writer's 
opinion, have any advantage over the above-mentioned apparatus as the prin- 
ciple is the same, although the urine must be diluted. 

Robert's Method. 

This method has been recommended for the quantitative determination 
of sugar and is based upon the fact that the specific gravity of the urine is 
changed in a quantitative way when the sugar of the urine is fermented. 



Fig. 87. — Lohn- 
stein 's fermenta- 
tion tube for undi- 
luted urine. 




Fig. 88. — Lohnstein's fermentation 
tube for diluted urine. 



The urine must be acid before applying this test. A piece of yeast about 
the size of a bean is added to the urine which is allowed to ferment at incubator 
temperature until no further qualitative test for sugar is obtained. This will 
usually require from 24 to 48 hours, so that a trace of sodium fluorid should 
be added to the urine to prevent bacterial action. The specific gravity of the 
urine before it is subjected to fermentation is very carefully taken either with 
a very accurately standardized hydrometer or, preferably, with the pycnometer. 
After fermentation is complete the specific gravity of the fermented urine is 
determined in the same way. The difference in the specific gravity of the 
two specimens is then multiplied by 234 to obtain the percentage of sugar. Or, 



THE URINE. 305 

according to Purdy, each degree of specific gravity lost in fermentation corre- 
sponds to 1 grain of sugar per fluidounce. 

If the specific gravity be accurately determined the results are correct 
within 0.1 per cent. As this method requires, however, the use of a pycnometer 
and a very accurate chemical balance, it can hardly be recommended to the 
general practitioner for his work. 

(6). Levulose (d -fructose), CH 2 OH— (CHOH) 3 — CO— CH 2 OH. 

Levulose is found very widely distributed throughout the vegetable king- 
dom, especially in fruits. Honey is almost a pure levulose. It may be found 
in the urine, transudates, or exudates, after a large intake of levulose-containing 
food or may occur spontaneously, when the subject has taken little such food. 
The levulosuria, reported by Zimmer, Ventzke, Czapek, Worm-Miiller, Seegen, 
Mauthner, Cotton, Roehmann, Personne, Henninger, Marie, and Robinson, 
must be accepted with reserve, as the incompleteness of the methods then in use 
afforded no certain means of recognizing levulose (Neuberg 1 ). The most 
authentic cases of true levulosuria are those of May, Schlesinger, Rosin and 
Laband, Lepine and Boulud, and Neubauer. This pure levulosuria occurs 
in both sexes and at all ages, the amount of sugar excreted being subject to 
variations from 2.7 grams per diem (Schlesinger) to 24 grams (Lepine). 

More common than pure levulosuria is its association with a glycosuria. 
This combination appears in all forms of diabetes, in the severe types levulose 
being practically never missed, according to Umber, especially when no restric- 
tion is placed on the carbohydrate intake. Neubauer's observations on this 
point are interesting. He finds that withdrawal of carbohydrates causes both 
levulose and dextrose to disappear from the urine. When levulose was given 
it was utilized, but when glucose was taken it was less completely assimilated, 
being excreted in part as levulose. 

The tolerance for levulose is, as a rule, less than that for glucose, so that 
we are not surprised to find that the administration of 100 grams of levulose 
to normal individuals is followed by a levulosuria in about 10 per cent, of cases, 
while no such effect may be observed in diabetes mellitus. Here the question of 
individual tolerance must be considered, as Umber finds 25 grams of levulose 
excreted following an intake of 100 grams, while the writer has observed an 
excretion of 75 grams on the same intake. Strauss 2 finds that an alimentary 
levulosuria occurs, after an intake of 100 grams of levulose, in 90 per cent, of 
cases of functional hepatic disturbance. This test would seem, therefore, to 
be a valuable indication of hepatic insufficiency, although it is not pathogno- 
monic. Also alimentary galactosuria, following the administration of 40 grams of 
galactose to the fasting stomach, as originated by Bauer, 3 has been shown by 
Reiss and Jehn and, also by Roubitschek to be inconstant. A decidedly 

1 Handbuch der Pathologie des Stoffwechsels, Berlin, 1907, 716; also, Cammidge and 
Howard, Lancet, 1915/I, 320. 

2 Deutsch. med. Wchnschr., 1901, XXVII, 757 and 786; Ibid., 19 13, XXXIX, 1780. See, also 
Oszacki and Wagner, Med. Klin., 1913, IX, 1549; Draudt, Arch. f. exper. Path. u. Pharmakol., 
1913, LXXII, 457; Hohlweg, Munch, med. Wchnschr., 1913, LX, 2271; and Schirokauer, 
Ztschr. f. klin. Med., 1913, LXXVIII, 462. 

3 Deutsch. med. Wchnschr., 1908, XXXIV, 1505. 

20 



306 DIAGNOSTIC METHODS. 

positive excretion of galactose is observed only in cases in which the entire 
liver parenchyma is presumably involved, as in the acute infections and intoxi- 
cations characterized by catarrhal jaundice. Cases of cirrhosis, carcinoma, 
cholelithiasis and phosphorus poisoning react either negatively or very slightly. l 

The chemical reactions of levulose are very similar to those of dextrose. 
Owing to the presence of the ketone group in its molecule, it shows the same 
reducing actions as does the aldehyd group of the glucose. Like glucose it 
ferments, but not quite so readily. It is levorotatory, its specific rotation 
being (a) D = —91 degrees. Leo has reported the finding of a levogyrate car- 
bohydrate in diabetic urines, which he believes to be laiose. This differs from 
levulose in being unfermentable with yeast. Levulose forms exactly the same 
phenylosazon as does glucose, so that it is a matter of great difficulty to differ- 
entiate these bodies by the tests given above. Lobry de Bruin and Alberda von 
Eckenstein have shown that glucose and levulose may change, the one into the 
other, by the action of traces of alkali, acids, and neutral salts, such as sodium 
acetate. 

Seliwanoff's Test. 

This test has been advanced as one characteristic for the ketoses in distinc- 
tion from the aldoses. Ten c.c. of urine are treated with a few crystals of resor- 
cin and 5 c.c. of concentrated HC1. If the mixture be warmed a brilliant red 
color appears in the presence of a ketone (levulose) while no coloration is ob- 
served with an aldehyd (glucose) . 2 Miiller has shown that glucosamin gives this 
test, while R. and O. Adler find a reaction in the presence of nitrous acid. 
If the mixture be heated too strongly or too long, mannose and maltose may 
also give a positive test. Adler finds that the use of acetic acid with a trace 
of HC1 gives better results than HC1 alone. It will be seen, therefore, that this 
test is not so characteristic as was believed, but it serves to distinguish levulose 
from glucose, which is the important point. 

If the red solution formed in this reaction be neutralized with sodium 
carbonate and extracted with amyl alcohol or, preferably, with acetic ether 
(Borchardt), the extract will have a yellow color with a faint green fluores- 
cence and becomes rose-red on the addition of alcohol. The spectrum of this 
solution shows a sharp line in the green between E and b, while if the solution 
be quite concentrated a second weaker line will be seen in the blue at F. 
Methyl-phenylhydrazin Test. 

Neuberg 3 has definitely shown that fructose forms a characteristic osazone 

1 Reiss and Jehn, Deutsch. Arch. f. klin. Med., 1912, CVTII, 187; Roubitschek, Ibid., 226; 
Foster, Am. Jour. Med. Sc, 1912, CXLIII, 830; Editorial, Jour. Am. Med. Assn., 1913, LX, 
287; Bloomfield and Hurwitz, Bull. Johns Hopkins Hosp., 19 13, XXIV, 375; Hurwitz and 
Bloomfield, Ibid., 380; Isaac, Ztschr. f. physiol. Chem., 1914, LXXXTX, 78; Wagner, Ztschr. 
f. klin. Med., 1914, LXXX, 174; Hatiegan, Wien. klin. Wchnschr., 1914, XXVII, 358; Fleck- 
seder, Ibid., 475; Arai, Deutsch. med. Wchnschr., 19 14, XL, 792; Worner and Reiss, Ibid., 
907; Maliwa, Med. klin., 1914, X, 762; Friedman and Strouse, Arch. Int. Med., 1914, XIV, 
531; Hoffmann, Ztschr. f. exper. Path. u.Therap., 19 14, XVI, 337; Schede, Jahrb. f . Kinderhkde., 
1 91 5, LXXXII, 45. 

2 Konigsfeld (Biochem. Ztschr., 19 12, XXXVIII, 310) shows that the HC1 must not have 
a final concentration greater than 12.5 per cent, and that the heating should not be longer than 
20 to 30 seconds. If dextrose, also, be present in amount greater than 2 per cent., the possibility 
of its conversion into levulose must be remembered. See, also, Jolles, Ibid., 1912, XLI, 331. 

3 Ztschr. f. physiol. Chem., 1902, XXXVI, 227; Ber. d. d. chem. Ges., 1902, XXXV, 959; 
Ibid., 1904, XXXVII, 4616. 



THE URINE. 307 

with methylphenylhydrazin, while no such compound is obtained with glucose 
mannose, or glucosamine. The test is, therefore, the most reliable and scien- 
tific one for the presence of levulose, although it is not clinically so acceptable 
as the Seliwanoff reaction. 

The formation of this osazone occurs according to the following equation. 

CH 2 OH-(CHOH) 3 -CO-CH 2 OH + 2NH 2 -N(CH 3 )(C 6 H s ) = CH 2 OH- 
(CHOH) 3 -C CH + 2H.O + 2H 

II II 

(CH 3 )(C 6 H 5 )N-N N-N(CH 3 )(C 6 H 5 ) 

Technic. 

The urine is acidified with acetic acid and boiled to remove albumin if 
present. The mixture is then filtered and the clear filtrate, which must be 
acid, is evaporated in a vacuum, at a temperature not over'40 , to a thin syrup. 
The reaction must remain acid during the evaporation so that a drop of 
acetic acid may be added if necessary. The residue is thoroughly extracted 
with 98 per cent, alcohol, using an amount of alcohol equal to one-half 
the original volume of urine. Filter and re-extract the residue with alcohol 
should any reducing action be observed in it. The alcoholic extracts are mixed 
and decolorized with animal charcoal. A portion of the extract is examined 
for its sugar-content by Fehling's test, all of the reduction being attributed to 
levulose. Methylphenylhydrazin is then added to the alcoholic solution, 
which should not measure over 30 c.c. The amount of the hydrazin to be 
added is in the proportion of 3 molecules for 1 of levulose or, in other words, 
for each gram of sugar a trifle over 2 grams of methylphenylhydrazin. The 
mixture is allowed to stand for a few hours in the cold and filtered if a precipi- 
tate forms. The filtrate is treated with 50 per cent, acetic acid, using the same 
amount of acid as of the methylphenylhydrazin, and sufficient alcohol is 
added to give a clear solution. The mixture is heated from three to five min- 
utes or, preferably, allowed to stand at 40 C. for 24 hours in an incubator. 
Crystals will usually separate out at the end of this time, but if not they will 
appear on the addition of a few drops of water. The crude product is purified 
by recrystallization either from a mixture of chloroform and petroleum ether or 
from hot water to which pyridin has been added. The yield by this method 
is 81 per cent, of the total sugar if pure solutions are used, while from urine it 
is but 50 per cent. 

The crystals of methylphenylosazone are delicate yellow, long, fine, needles 
melting from 158 to 160 C. This method can hardly find application in 
the hands of the general practitioner. 

Pure levulosuria is recognized by the levorotation of the urine, which 
possesses reducing properties and is capable of fermenting with yeast. After 
fermentation, the urine loses its reducing and optical properties. The presence 
of a levulosuria is indicated by a considerable difference between the results 
obtained by titration and polarization, providing a glycosuria be coexistent. 
The Seliwanoff reaction should be used as a routine in every case which shows 
both fermentation and reduction. The general practitioner will rarely have 



308 DIAGNOSTIC METHODS. 

access to a polariscope and even then may be misled if other interfering sub- 
stances, such as albumin, glycuronic acid, and /3-oxybutyric acid be present. 
Do not presuppose that glucose is the only fermentable and reducing sugar of 
the urine, as the recognition of the true condition may make much difference 
in the treatment as well as in the prognosis. 

(c). Pentose. 

The pentose 1 group of carbohydrates comprises eight possible stereo- 
isomers with the general molecular formula of C 5 H 10 O 5 . Three members 
of this group, rhamnose, fucose, and chinovose, are substituted pentoses, viz., 
methyl pentoses having the formula C 6 H 12 5 . These latter are the exceptions 
to the general rule that carbohydrates contain the same number of carbon 
atoms as of oxygen atoms, and have led to the more scientific method of 
classifying the carbohydrates according to the number of oxygen atoms rather 
than of carbon atoms. The pentoses are widely spread throughout the vege- 
table kingdom in the form of their anhydrids or in combination with other 
groups of atoms. They are also found as constituents of the nucleoproteins 
of animal tissues, being especially abundant in the pancreas. 

It has been found that the ingestion of large amounts of pentose-contain- 
ing food, such as apples, cherries, plums, beets, and the leguminous vegetables, 
leads to the excretion of pentose in the urine. This alimentary type of pen- 
tosuria is characterized by the presence of optically active xylose or arabinose 
and appears after the ingestion of small amounts, in some cases following an 
intake of as low as 50 mg. of the pure carbohydrate. In true diabetes mel- 
litus the urine frequently contains 1-xylose which probably arises from the 
breaking down of the pancreatic nucleoprotein. Alfthan states that the 
pentoses are constantly present in diabetic urine, so that it is highly probable 
that these sugars might be found whenever search was made for them in diabetic 
conditions. It is not strange that these carbohydrates are not more frequently 
reported, as their reducing action leads to confusion unless controlled by fer- 
mentation methods. The excretion of pentoses in diabetic conditions is not 
necessarily increased in direct proportion to the intake, as their absorption may 
be so slow that accumulation is not possible before oxidation has occurred. 

Salkowski and Jastrowitz 2 reported in 1892 the finding of a pentosuria 
which was not associated with intake of pentose food nor with diabetes melli- 
tns. This type is known as idiopathic, essential, or intrinsic pentosuria, of which 
24 cases were found by Janeway 3 up to 1906. The peculiar thing of this type 
of pentosuria is that the sugar excreted is r-arabinose, an optically inactive pen- 
tose. This is the single exception in which an optically inactive pentose is 
found in all nature. This fact characterizes this type of pentosuria as an anom- 
aly of metabolism sui generis. The origin of this urinary pentose is still 
unsettled. The source must be within the organism, as no inactive arabinose 
is taken as food, and if it be given in experimental cases it appears in the urine 
as d-arabinose. Moreover, it cannot be derived from the nucleoprotein as the 

1 See Bendix, Die Pentosuria, Stuttgart, 1903. 
2 Centralbl. f. d. med. Wissensch., 1892, 337. 
3 Am. Jour. Med. Sc, 1906, CXXXII, 423. 



THE URINE. 309 

pentose in these cases is 1-xylose. 1 Neuberg suggests that galactose might be 
considered the source of this pentose, but no proof of this has been forthcoming. 

In the true idiopathic pentosuria the assimilation of other carbohydrates 
is unchanged and does not influence in any way the excretion of r-arabinose, 
although the active types of this pentose may be excreted at the same time 
in the urine. It is interesting to find that the pentoses taken in as food are 
excreted in different proportions by the diabetic and nondiabetic subjects. 
Thus von Jaksch observes that diabetics excrete from 49 to 82 per cent, of 
arabinose of the food and nondiabetics 1 to 47 per cent., while nondiabetics 
excrete from 19 to 55 per cent, of xylose and diabetics only a trace. The 
amount of pentose excreted in essential pentosuria has been reported as vary- 
ing between 0.08 and 1 per cent. Neuberg has recently shown that a certain 
amount of the r-arabinose is combined with urea in the form of a ureid, which 
does not reduce Fehling's solution until it undergoes hydrolysis with acid. For 
this reason he believes that the amount of pentose reported is in practically 
all cases 100 per cent, too low. Luzzatto 2 has reported the excretion of the 
optically active 1-arabinose entirely independent of the food intake. 

The pentoses reduce copper solutions as do other carbohydrates, but the 
reduction is much slower, appearing during the cooling of the fluid. Ten c.c, 
of Fehling's solution are reduced by 0.0542 gram of pentose. They do not fer- 
ment with yeast and do not give a typical reduction with the Almen-Nylander 
test, the color being a gray rather than a black. In the true iodiopathic pen- 
tosuria no reaction is observed with the polariscope, while in the alimentary 
type a slight dextrorotation is usually noted, although von Jaksch reports the 
excretion of an inactive arabinose after the ingestion of active pentoses. These 
pentoses form more or less typical osazones which melt between 157 and 160 , 
but the reaction is not so easily produced. 3 These pentosazones are readily 
soluble in warm water and show dextrorotation. The most characteristic 
chemical property of these types of carbohydrate is the formation of furfurol 
(C 5 H 4 2 ) when they are distilled in the presence of acids. The color reactions 
given below are based upon the production of furfurol and the formation of 
distinct colorations on treatment with various reagents. 

Tollen's Test. 
A few c.c. of concentrated hydrochloric acid are saturated with phloro- 
glucin, care being taken to leave a small amount undissolved. This solution 
is then divided into two equal parts, to one of which is added 1/2 c.c. of the 
suspected urine and to the other 1/2 c.c. of normal urine. Both tubes are then 
placed in a boiling water-bath for a few minutes, when an intense red zone 
will appear in the upper portion of the tube if pentose be present. After a few 
moments this red color will gradually spread throughout the fluid, while the 
control urine shows no marked change in color. It is advisable to remove 

1 Elliott and Raper (Jour. Biol. Chem., 1912, XI, 211; Ibid., 1913, XV, 481) suggest the 
possibility of this sugar being ribose. 

2 Beitr. z. chem. Physiol, u. Path., 1905, VI, 87. 

3 Klercker, Deutsch. Arch. f. klin. Med., 1912, CVIII, 277; Levene and La Forge, Jour. 
Biol. Chem., 1913, XV, 481.; Ibid., 1914, XVIII, 319; Rosenbloom, Jour. Am. Med. Assn., 
1 91 5, LXEV, 508. 



310 DIAGNOSTIC METHODS. 

the tubes from the water-bath as soon as the color appears, as the clearness 
of the reaction is interfered with by prolonged heating. The coloring matter 
is then extracted by shaking with amyl alcohol, when spectroscopic examina- 
tion will show an absorption band between D and E. 

This test reacts in the same way with glycuronic acid so that it has little 
value in differentiating pentose from the former substance. As a rule, the free 
glycuronic acid is not so easily split from its conjugated compound as is furfurol 
from pentose, so that the test is at least suggestive of pentose. 

Orcin Test. 

For this test the urine should be decolorized by heating with animal char- 
coal and filtering. Five c.c. of urine are treated with an equal volume of con- 
centrated hydrochloric acid and a few crystals of orcin are added. The mix- 
ture is then warmed approximately to the boiling-point, when a dark green 
color appears in the presence of pentose or glycuronic acid. The formation 
of a greenish-blue precipitate is very strong evidence of pentose rather than 
glycuronic acid. The pigment is then extracted with amyl alcohol, when spec- 
troscopic examination shows a characteristic absorption band between C and 
D. The presence of glucose may interfere with the reaction, so that it may be 
necessary to remove it by fermentation. 

Bial has modified this test in such a way that glycuronic acid is less apt 
to be a disturbing factor. His reagent consists of 500 c.c. of 30 per cent. HC1 
to which are added 1 gram of orcin and 25 drops of 10 per cent, ferric chlorid 
solution. Four to five c.c. of this reagent are heated to boiling and removed 
from the flame. The suspected urine is then added drop by drop, not exceed- 
ing 1 c.c. in all, when a green color should appear almost immediately if pen- 
tose be present. The heat employed is hardly sufficient to split off glycuronic 
acid. Here also glucose, if present, should be removed by fermentation with 
a pure culture of yeast rather than with compressed yeast, as the bacteria 
possibly present in yeast may break up the pentoses at the same time. 

The osazone may be formed as previously given under Glucose. The 
glucosazone is separated from the pentosazone by digesting with water not over 
6o° in temperature, the pentosazones being dissolved. If the pentosazone 
be treated with 20 c.c. of water and 5 c.c. of concentrated hydrochloric acid and 
distilled, the distillate will give a beautiful test with Bial's reagent, which 
absolutely eliminates glycuronic acid and other interfering substances possibly 
present in diabetic urine. 

Quantitative Determination. 

Neuberg and Wohlgemuth 1 have introduced a method by which the ara- 
binose of the urine may be accurately determined. A preliminary determina- 
tion of the sugar present is made by Purdy's solution, eliminating glucose 
by previous fermentation. If less than 1 per cent, of reducing sugar, which is 
assumed to be arabinose, is present, the urine must be concentrated in a vacuum 
so that the sugar content is slightly over 1 per cent. 

1 Ztschr. E. physiol. Chem., 1902, XXXV, 31 and 41. 



THE URINE. 311 

Technic. 

One hundred c.c. of urine are acidified with two drops of 30 per cent, 
acetic acid and evaporated on a water-bath to approximately 40 c.c. It is 
then treated with 40 c.c. of 96 per cent, alcohol, the mixture is allowed to stand 
for two hours, and is then filtered from the separated urates and inorganic 
salts. The residue is carefully washed with 40 c.c. of 50 per cent, alcohol. 
To the filtrate 1.4 grams of pure diphenylhydrazin are added and the mix- 
ture heated on a boiling water-bath for one-half hour, the alcohol being replaced 
as it evaporates. The mixture is allowed to stand for 24 hours and is filtered 
through a Gooch filter, using the mother liquor to transfer the precipitate. 
The crystals are then washed with 30 c.c. of 30 per cent, alcohol, and the 
Gooch with its contents dried at 8o° C. to constant weight. The amount of 
arabinose is obtained by multiplying the weight of the diphenylhydrazone by 
0.4747 or by dividing by 2.107. 

Cammidge's Reaction. 

Cammidge 1 has found that the urine in cases of pancreatic disease contains 
a substance which gives an osazone when treated with phenylhydrazin. In 
his earlier work* he was led to believe that this substance was possibly glycerin 
or a derivative. He advanced two reactions, the first of which he found to be 
due to a mixture of glycuronic acid and a true carbohydrate, while the second 
was apparently due only to the glycuronic acid. In his later studies on this 
reaction he was led to believe that the mother substance of the osazone was a 
pentose (probably xylose) split off from a dextrin-like body by the hydrolysis 
with hydrochloric acid. This view has been somewhat substantiated by others. 
The careful work done in recent years by Kinney, 2 Whipple and King, 3 Whipple, 
Chaffer and Fischer, 4 Swan and Gilb ridge, 5 Wilson, 6 and Roper and Stillman 7 
has proven conclusively that, although this reaction is given in some cases of 
pancreatitis, it is not pathognomonic as it occurs in many other conditions and 
may not, even, be present in pancreatic disease. 8 This test is not to be regarded, 
therefore, as of much diagnostic value. 

Technic. 

The writer gives only the improved method known as " Reaction C" refer- 
ring to the original work of Cammidge for reactions A and B. The urine to be 
tested should be a portion of the 24-hour specimen and must be freed from 
glucose and albumin by methods previously outlined. 

Forty c.c. of clear, filtered acid urine are acidified with 2 c.c. of concen- 
trated HC1 and boiled for 10 minutes. The mixture is then cooled and made 

1 Lancet, 1904, 1, 782; Ibid., 1905, II, 14; Robson and Cammidge, Surgery of the Pancreas, 
London, 1907. See Cammidge and Howard, Lancet, 1914, II, 791; Pekelhering and van 
Hoogenhuyze, Ztschr. f. physiol. Chem., 1914, XCI, 151; Lameris and van Hoogenhuyze, 
Nederl. Tydschr. v. Geneesk., 1914, II, 675. 

2 Am. Jour. Med. Sc, 1910, CXL, 878. 

3 Bull. Johns Hopkins Hosp., 1910, XXI, 196. 

4 Ibid., 1910, XXI, 339. 

6 New York Med. Jour., 19 10, XCI, 781. 

6 Trans. Assoc. Amer. Phys., 1910. 

7 Arch. Int. Med., i9ii,VII, 252. 

8 See Mayesima, Mitt. a. d. Grenzgeb. d. Med. u. Chir., 1912, XXV, 403; also, Karas, 
Ztschr. f. klin. Med., 1913, LXXVII, 125. 



312 DIAGNOSTIC METHODS. 

up to 40 c.c. with distilled water. The excess of acid is then neutralized 
by the addition of 8 grams of lead carbonate and the mixture cooled if necessary. 
Filter off the resulting precipitate and treat the nitrate with 8 grams of powdered 
tribasic lead acetate to remove the glycuronic acid. Filter, treat nitrate with 
4 grams of powdered sodium sulphate, heat to the boiling-point, and allow to 
cool. The lead sulphate is removed by nitration. Ten c.c. of the clear 
filtrate are made up to 17 c.c. with distilled water, 0.8 gram of phenylhydrazin, 2 
grams of sodium acetate, and 1 c.c. of 50 per cent, acetic acid are added and 
the mixture boiled for 10 minutes. Filter while hot and make the filtrate 
up to 15 c.c. with warm water. The mixture is allowed to cool, when yellow 
crystals arranged in sheaves and rosettes may be observed under the high- 
power lens. 

(d). Lactose (C 12 H 22 O u ). 

Lactose is found in the urine of women during the period of lactation 
and may be found in patients who have been on an exclusive milk diet for a 
long period. A distinct type of alimentary lactosuria is observed on account 
of the low assimilation limit for milk-sugar. In breast-fed children with 
gastrointestinal disturbance lactose associated with galactose may be found 
in the urine. In this case Langstein and Steinitz 1 have shown that the excretion 
is not due to failure of the normal enzyme, but to an unknown derangement 
of the activity of lactase, which renders it incapable of splitting up the whole 
of the lactose, the remainder being absorbed unchanged. From the portion 
which is split up in the bowel the resulting easily assimilable glucose is utilized 
by the organism, while galactose partly escapes by the kidneys, on account 
of the much lower assimilation limit for this latter carbohydrate. Lactose 
in these cases is usually associated, therefore, with galactose (Neuberg). 
According to the work of Voit, an increase of lactose in the diet of a diabetic 
is associated with an increased output of glucose. 

The usual form of lactosuria is that observed in the parturient female. 
It is ordinarily first seen a few days after delivery of the child, but occasionally 
appears during the latter days of gestation, as Ney, Lemaire, and Porcher have 
shown. The amount of lactose excreted by the nursing mother equals 2 to 3 
per cent., according to Naunyn, while McCann places the average at 0.35 
per cent, for the first few days of the puerperium. Lactose may continue in 
the urine for some time, the actual amount depending upon the quantity of 
milk as well as its quality. 2 If nursing is interrupted for any reason, more 
lactose will be found than when nursing is regular. 

Lactose reduces copper solutions, although somewhat less actively than 
does glucose. It also shows a positive Almen-Nylander reaction. It has a 
strong rotatory power, its specific rotation being practically the same as that 
of glucose ( + 52.5 ). It does not ferment with yeast, although bacteria if present 
may hydrolyze it into its constituents, glucose and galactose, the former of which 
will show fermentation. It is, therefore, advisable when applying the fermen- 

1 Beitr. z. chem. Physiol, u. Path., 1906, VII, 575. 

2 See Gronvall, Biochem. Ztschr., 1912, XL, 145; Rosenbloom (Jour. Am. Med. Assn., 
1 91 5, LXIV, 508) reports the finding of lactose in the urine of an adult male after intake of 
milk. 



THE URINE. 



3*3 



tation test not to judge of a reaction which has progressed longer than a few 
hours. With phenylhydrazin it forms a lactosazon which appears in the form 
of sheaves of delicate curved needles much resembling bunches of yellow thread. 
These crystals melt at 200 C. The test is not easily obtained unless the urine 
be concentrated to a small bulk and the residue extracted with alcohol, when 
the alcohol is evaporated and this residue taken up with water and the phenyl- 
hydrazin test then applied. 
Rubner's Test. 

Ten c.c. of urine are treated with an excess (3 grams) of lead acetate 
and boiled for a few minutes. 1 The yellowish or brown solution is then filtered 
and ammonia added to the filtrate until a slight permanent precipitate remains. 
An intense brick-red fluid is obtained which later shows the deposition of a 
cherry-red precipitate with a colorless supernatant fluid. This test is not very 
delicate as it shows lactose only when present in amounts varying from 0.3 
to 0.5 per cent. Glucose gives with this test a red solution, but a more distinctly 
yellow precipitate. 

Lactosuria is to be assumed when the urine possesses reducing properties 
and dextrorotation, but is incapable of fermenting with ordinary yeast within 
1 2 hours. If the urine be boiled with 2 per cent, sulphuric acid and then neutral- 
ized, its optical activity will be increased and it will be capable of undergoing 
fermentation. It is to be remembered in testing for the amount of lactose by 
the use of Fehling's solution that 10 c.c. of this solution are reduced by 0.0678 
gram of lactose instead of by 0.05 as in the case of glucose or levulose. 

(e). Maltose (C l2 H 22 O n ). 

Maltose has occasionally been reported in the urine, although many of the 
cases are questionable as the proper identification of the sugar was not 
thoroughly carried out. The most reliable cases appear to be those of Noble, 
von Ackeren, Rosenheim and Flatow, and especially that of Magnus-Levy. 
In this latter case the urine showed a considerable excess of rotation when 
compared with its reducing power. After inversion with dilute acid, by 
which each molecule of maltose was converted into two molecules of glucose, 
the rotation diminished and the reduction increased, so that the polarimetric 
and titration methods gave concordant results. The urine underwent complete 
fermentation with synchronous loss of optical activity and of reducing power. 
Calculations founded on these determinations showed that 1.5 per cent, of 
maltose and 2 per cent, of glucose were present. This seems to be a case in 
which the amount of maltose excreted exceeds all records (Neuberg) . The cases 
in which maltose appears in the urine seem to be those of disease of the pancreas, 
especially those with interstitial lesions. 

Maltose reduces copper solutions, but not as strongly as does glucose. 
Ten c.c. of Fehling's solution are completely reduced by 0.0807 gram of maltose. 
It is much more strongly dextrorotatory than glucose and forms an osazone 
which crystallizes in large prism-like needles arranged in sheaves, and melts 
at 207 C. This osazone is soluble in water and shows a dextrorotation, it being 
more distinctly identified by determination of the nitrogen content, which 
1 See Cole, Biochem, Jour., 1914, VIII, 134; also, Rogerson, Ibid., 1915, IX, 245. 



314 DIAGNOSTIC METHODS. 

should equal 10.6 per cent. Maltose ferments with yeast only after inversion 
by heating with acid, the splitting products being two molecules of glucose. 

Other carbohydrates, such as dextrin, isomaltose, and saccharose, have 
been reported in the urine. These are extremely rare and need little comment 
in this place. In the case of cane-sugar the assimilation limit is so high that 
an alimentary saccharosuria could occur only after an enormous intake. 
Spontaneous excretion of cane-sugar has never been actually proven, but 
this sugar may be found in the urine of hysterical patients who have added 
it to deceive the physician. The so-called animal gum, first isolated by Land- 
wehr, seems to be a normal constituent of urine. Alfthan 1 finds it is present 
in practically every case of diabetes to the extent of 1 to 37 grams per diem. 
This substance is probably not a definite chemical body, but a mixture of 
several. Little is known of its chemistry. 

Inosite was regarded for a long time as a carbohydrate, but it is now 
known to be a hexaoxyhexahydrobenzol with the formula C 6 He(OH) 6 . This 
substance, has, therefore, nothing to do with true carbohydrate metabolism, 
but is discussed at this point as it has so long been regarded in this connection. 
Inosite enters into the composition of almost all animal tissues and occurs 
both in the optically active and inactive forms. A physiologic excretion of 
inosite is not infrequent, according to Hoppe-Seyler. It may occur in nephritis, 
diabetes mellitus and insipidus, and after a large intake of animal food. 

(/). Glycuronic Acid (CHO- (CHOH) 4 -COOH). 

Glycuronic acid is an intermediate product of the oxidation of carbo- 
hydrate, the CH 2 OH group being converted into CHO while the original 
CHO group is oxidized into CO OH. This acid still retains the aldehyd 
group, in consequence of which it shows the same reducing action as does 
glucose. It seems to be characteristic of glycuronic acid that, when pro- 
duced naturally, it is never found in the free state, but only in the combined 
form as the conjugated glycuronic acid. It seems to be especially capable 
of combining with substances showing alcoholic or phenolic characteristics. 2 
The free glycuronic acid may be split off from its conjugated compounds 
by heating with acid and other hydrolyzing agents. The conjugated glycuro- 
nates are levorotatory while the free acid shows dextrorotation. Among 
the substances with which glycuronic acid combines we find chloral hydrate 
butyl chloral hydrate, chloralamid, camphor, menthol, carbolic acid, resorcin, 
acetanilid, antipyrin, phenacetin, pyramidon, sandal-oil, morphin and 
cocain. The normal metabolism following intake of any of these substances 
is such that excretion of conjugated glycuronic acids will follow and may, 
therefore, lead to the assumption of sugar in the urine unless precautions are 
taken properly to differentiate these compounds. Besides conjugated gly- 
curonic acid of the above type we find a combination of urea with glycuronic 
acid as well as a certain amount of indoxyl, skatoxyl, phenol, and cresol in 
combination with this acid. Most of the products of bacterial decomposition 

1 Ueber dextrinartige Substanzen in diabetischen Harn, Helsingfors, 1904. Cainmidge 
and Howard, Lancet, 1914, II, 791. 

2 See Hamalainen, Skand. Arch. f. Physiol. 19 13, XXX, 196. 



THE URINE. 315 

in the intestine are excreted in combination with sulphuric acid, but some is in- 
variably present as a conjugated glycuronate. 

The origin and formation of glycuronic acid within the system is not 
entirely understood. It has been supposed to be derived from protein as 
especially advocated by Loewi, but Mayer has rather disproven Loewi's work, 
and shows that probably glycuronic acid is a direct derivative of glucose and 
that all carbohydrate oxidation must pass through the intermediate stage of 
glycuronic acid. 

The status of this question is very well summed up by Neuberg as follows : 
The formation of glycuronic acid out of protein is by no means excluded, nor 
yet from fat; but as it is difficult to eliminate the direct formation of glycuronic 
acid or its secondary development from previously existing grape sugar, 
Mayer justly contends that the question of glycuronic acid formation from 
these substances is practically included in the broader question of the formation 
of sugar from fat and protein. 

The exact point of conjugation of glycuronic acid is unsettled. It 
has been assumed by some to occur in the liver, while others find that the 
liver plays no part. It is probable that the synthesis takes place in various 
parts of the organism. 

It has been shown that the output of glycuronic acid may be increased 
in diabetes mellitus, in mild cases the unoxidized sugar being present largely 
in this form. Mayer 1 advances the hypothesis of incomplete oxidation of 
sugar to explain its appearance in these cases. 2 He shows that after the ad- 
ministration of glucose in amounts beyond the assimilation limit, an occasional 
excretion of glycuronic acid occurs with an equivalent diminution of the ethereal 
sulphates. It is possible that the substance conjugating with glycuronic 
acid is unknown and that we have the same results as though similar substances 
were introduced by mouth. As Mayer has advanced no direct proof of the 
correctness of his theory, the decision must be left for the future. Clinically, 
the question of the highest importance is whether the excretion of glycuronic 
acid is of any diagnostic value and whether it is of prognostic significance in 
diabetes. It does not seem wise to assume that a patient showing an occa- 
sional increase in the glycuronic acid excretion, which cannot be accounted 
for by intake or increased production of conjugating substances, will in the 
future show typical diabetes. Edsall does not believe in the value of glycuronic 
acid in the diagnosis of a latent diabetes, nor does Neuberg regard an increased 
excretion of glycuronic acid as the original derangement which may determine 
other deviations from health. 3 

Very few of the conjugated glycuronates show reducing action when 
treated with copper solutions using the precautions previously laid down. 
The chloral and camphor compounds are much more apt to produce typical 

Ztschr. f. physiol. Chem., 1901, XXXII, 518; Berl. klin. Wchnschr., 1903, XL, 292 
and 514. See Biberfeld, Biochem. Ztschr., 1914, LXV, 479; Roger and Chiray, Bull. 
l'Acad. de Med., 191 5, LXXIII, 444- 

2 See Conzen, Ztschr. f. klin. Med., 1912, LXXV, 426. 

8 See Medigreceanu (Jour. Exper. Med., 1913, XVIII, 259) for a discussion of the glycu- 
onic acid excretion in pneumococcus infections. 



316 DIAGNOSTIC METHODS. 

reduction, but even these require heating for somewhat longer periods than does 
glucose. A diagnosis of a glycuronic acid excretion is based upon the following 
points : The fresh urine is levorotatory, but shows little or no reducing prop- 
erties and does not ferment. This same finding will be observed if /3-oxybuty- 
ric acid be present so that a diagnosis may not rest on these findings alone. 
After being boiled with dilute acid for a period varying from one-quarter 
to three-quarters of an hour, the levorotation is changed to dextrorotation 
and the urine shows strong reducing powers. Such tests will not be given by 
/3-oxybutyric acid. In some cases after heating the urine with acid the action 
upon light may remain levorotatory or the solution may be optically inactive, 
on account of the fact that the conjugating substance may be levorotatory 
or that complete hydrolysis has not been effected. On heating the urine 
for some time with BiaFs modification of the orcin test a positive reaction 
appears with the liberated glycuronic acid. This acid crystallizes with phenyl- 
hydrazin forming distinct yellow needles which melt at 114 to 115 C. This 
test is, however, not readily obtained so that it is difficult to identify glycuronic 
acid by its osazone. It will be seen, therefore, that glycuronic acid is differen- 
tiated from the pentoses largely by its levorotation when in the conjugated 
state or dextrorotation when free. 

Tollens (Ber. d. d. chem. Ges., 1908, XLI, 1788) has advanced a test which 
permits of a clear differentiation between glycuronic acid and pentose. To 
5 c.c. of urine add 1/2 c.c. of a 1 per cent, alcoholic solution of naphthoresorcin 
and 5 c.c. of concentrated HO. Warm over the free flame to boiling or place 
in the boiling water-bath for 15 minutes. Allow to stand for 4 minutes and then 
cool under running water. Add an equal volume of ether and shake vigorously. 
On separating, the ether layer shows, in the presence of glycuronic acid, a 
violet to blue coloration. Spectroscopic examination reveals a sharp absorption 
band at the D line. The test is not given by pentose. (See Tollens, Ztschr. f . 
physiol. Chem., 1908, LVI, 115; Jolles, Ibid., i9i2,LXXXI, 203; also, Neuberg 
and Schewket, Bio chem. Ztschr., 1912, XL1Y, 502.) 

Golds chmiedt 1 has introduced a test which Mayerhofer 2 believes of great 
value to the pediatrician, as it indicates intestinal derangement better than 
indican. One-half to 1 c.c. of urine is treated with 2 drops of a 15 per cent, 
alcoholic solution of a-naphthol. Carefully overlay 3 to 4 c.c. of concentrated 
H 2 S0 4 with this mixture. A violet ring is observed, which changes to a dis- 
tinct emerald green on standing. In this test the urine must be free from nitrites 
and nitrates. 

Neuberg's Test. 3 
Five hundred c.c. of urine are treated with sufficient sulphuric or phosphoric 
acid to make the acidity from 1 to 2 per cent. This acidified urine is then 
heated in an autoclave for two hours at a temperature of 115 C. The mixture 
is then cooled, neutralized with sodium carbonate, acidulated with acetic 
acid, and filtered. Two hundred and fifty c.c. of the filtrate are mixed with a 

1 Ztschr. f. physiol. Chem., 19 10, LXV, 389; 19 10, LXVII, 194. 

2 Ztschr. f. physiol. Chem., 1911, LXX, 391. 

3 Ber. d. d. chem. Ges., 1910, XXXII, 2395; Ztschr. f. physiol. chem., 1905, XLIY, 127. 



THE URINE. 317 

hot aqueous solution of 5 grams of parabromphenylhydrazin hydrochlorate 
and 6 grams of sodium acetate. The mixture becomes cloudy at once, but on 
heating the cloudiness will disappear. As the mixture cools needle-shaped 
crystals will separate out and may be filtered off, the filtrate being again heated 
and cooled to obtain more crystals. This process may be repeated until 
no more crystals form. These crystals are then washed with distilled water 
followed by absolute alcohol, and are then recrystallized by dissolving in 60 
per cent, alcohol and gradually evaporating. They are clear yellow in color, 
melt at 236 C, and show marked levorotation when dissolved in a mixture 
of pyridin (4) and absolute alcohol (6). This test is distinctive. 

(3). Acetone Bodies. 

By the acetone bodies 1 we mean acetone, diacetic acid and i3-oxybutyric 
acid. The latter of these is the mother substance so that this group would 
better be called the /3-oxybutyric bodies. The chemical relation between 
these bodies is very close, the /?-oxybutyric acid being oxidized to diacetic 
acid, which then splits up into acetone and carbon-dioxid. This may be 
seen from the following formulas : 

CH 3 -CHOH -CH 2 -COOH /3-oxybutyric acid. 
CH 3 -CO -CH 2 -COOH Diacetic acid. 

CH 3 -CO -CH 3 Acetone. 

Formerly these substances were supposed to be derived from protein material, 
the /3-oxybutyric acid being formed from the /?-amino acids by desamidization 
and oxidation in the /? position. This theory is, however, not generally held 
at present, being replaced by the more modern idea that the fats are the chief 
source of the acetone bodies. It has been found that in perfectly sound, 
well-nourished individuals the addition of fat causes only a very slight increase 
in the output of acetone bodies and, strangely enough, that butyric acid itself 
causes no acetonuria. On the other hand in normal individuals from whom 
the dietary carbohydrate has been removed or in a diabetic who is not utilizing 
what carbohydrate he may be allowed, a marked excretion of acetone bodies 
may occur. While a portion of this acetone may possibly be derived from 
the carbohydrate groups of the protein molecules, it can hardly explain the 
enormous excretion in diabetes, as the amount of protein catabolism, as shown 
by the urinary nitrogen, is greatly insufficient to yield any such amount of acetone 
bodies. The fats are, therefore, the more probable source of these bodies. 
As long as the body is supplied sufficient carbohydrate or is able to oxidize 
a sufficient amount the acetone bodies of the urine remain low; but when the 
system is no longer capable of oxidizing the carbohydrates, the amount of 
acetone bodies increases to a marked extent. We see, therefore, that the older 
method of allowing diabetics practically no carbohydrate food may directly 
lead to aggravation of the symptoms which the withdrawal was supposed to 
remedy. In other words, the normal or the diabetic individual must have a 

x See Waldvogel, Die Acetonkorper, Stuttgart, 1903; Magnus-Levy, Ergebnisse der klin. 
Med., Jena, 1908; Lepine, Rev. de Med., 1913, XXXIII, 601; Marriott, Jour. Biol. Chem., 
1914, XVIII, 241. • 



318 DIAGNOSTIC METHODS. 

certain amount of carbohydrate food in order that proper metabolism may 
be maintained. 1 This is not the time or place to discuss the therapy or dietetic 
treatment of diabetes, but it must be remembered that the most successful 
diet is one which contains carbohydrates up to the point of tolerance. Even 
here we find that certain types of carbohydrates may be given to diabetics 
without increasing the glycosuria, while at the same time leading to a dimi- 
nution in the excretion of acetone bodies. 

The condition arising from a surcharging of the blood with these acetone 
bodies is known as acidosis. 2 For a long time it was supposed that the carbo- 
hydrates were not only accountable for glycosuria, but also for the acetonuria 
and acidosis noted in diabetes. In the advance of pathologic chemistry it 
has been shown that, instead of causing these latter symptoms and conditions, 
the carbohydrates in reality lessened them. This may be shown by the 
administration of a definite amount of sugar, especially in the milder types of 
diabetes, to patients from whose diet sugars have been previously excluded. 
The omission of sugars from the diet forces the organism to utilize its protein 
and fat and thus gives rise to an accumulation of nitrogenous and fatty metabolic 
products as well as to an increase in the acids of the body fluids. In this 
way an acidosis already present would be increased in intensity. If the carbo- 
hydrate-free diet be continued for some time, a readjustment takes place and 
the acetonuria may gradually diminish, as is instanced by the fact that certain 
races show no acetonuria even though on an absolutely carbohydrate-free diet. 

With regard to the proteins as the mother substances of these bodies, we 
must admit that their influence is to some extent a double one. In the first 
place protein tends to diminish the acetonuria on account of its carbohydrate 
content, those proteins containing the greatest number of carbohydrate 
groups not necessarily exerting the greatest effect either on this condition or 
on the glycosuria. With a diet excessive in protein the influence is, however, 
not of this sort. The sulphuric and phosphoric acids as well as the small 
amount of acetone bodies formed by the hydrolysis of the protein tend to increase 
an existing acidosis, while the carbohydrates formed in the splitting of these 
proteins may greatly increase an existing glycosuria. These points, together 
with the fact that the products of nitrogenous metabolism may greatly increase 
the osmotic tension of the blood and thus lead to disordered cell function, 
show us that proteins cannot be advantageous as an exclusive diet in diabetes. 
As is well known, the nitrogenous excretion is much more marked in a diabetic 
than in a nondiabetic owing to several factors. In the first place, the diabetic 
consumes more protein than the normal individual because his diet is limited 
as regards carbohydrates and must be made up to a requisite caloric value by 
protein and fat. Secondly, owing to the lack of the protein-sparing function 
of the carbohydrates, excessive protein is broken down and elaborated in order 
to furnish a portion of the energy necessary to maintain the body function. It 

1 See Blodgett, New York, Med. Jour., 191 5, CII, 458. 

2 Folin and Denis, Jour. Biol. Chem., 1015, XXI, 183; Stern, Arch. Diag., 191 5, VIII, 128; 
Beddard. Pembrey and Spriggs, Brit. Med. Jour., 1915, II, 389. See Straub, Deutsch. Arch, 
f. klin. Med., 1913, CIX, 223; also, Benedict and Joslin, Deutsch. Arch. f. klin. Med., 1913, 
CXI, 333. 



THE URINE. 319 

must, however, be said that the diabetic protects himself for a time from the 
unusual loss of protein by the utilization of fat. 

Concerning the fats, it is to be recalled that, although formerly accredited 
with no power of influencing acetonuria, to-day they are regarded as directly 
affecting this condition to a great extent. This is true of the fatty acids, 
especially of the lower members, and not of the neutral fats. If the contention 
of Kastle and Loevenhart be true, that a reversible action of lipase converts 
the fatty acids and glycerin formed by a previous hydrolysis again into neutral 
fats, then the influence of fats on the acidosis is variable, or else we must assume 
a lack of lipase in the cells of the diabetic. We know that the fatty acids belong 
to the ketoplastic group (substances increasing excretion of acetone bodies), 
yet as Borchardt has recently shown this ketoplastic action is, doubtless, due 
to the union of the fatty acids with glycerin, thus withdrawing from the 
system the antiketoplastic body, glycerin, and enabling the remaining fatty 
acids to exert their influence on the formation of the acetone bodies. Fats 
do not increase an existing glycosuria as many experiments have shown, yet 
we must grant that a formation of sugar from fat does take place. Von Noorden 
speaks of a "facultative formation of sugar from fat," referring to the fact 
that the demand for sugar may become so great that this source is called upon 
to furnish its quota of carbohydrates. We must also remember that the syn- 
thesis of fat from disintegrated carbohydrate is much affected in diabetes. 
Were this not the case, a large part of the sugar, reaching the blood as such, 
would be synthesized by the fat-forming cells and glycosuria would be dimin- 
ished. Conceiving this latter function to be normal while the former is abnor- 
mal, we may readily see the close relationship between obesity and later diabetes. 
With an excessive diet of fat no more fat is oxidized than when the diet is low 
in fat. In the latter case, the body-fat is utilized to furnish the difference, while 
in the former the excess is deposited in the usual fat depositories. 

Besides the excretion of acetone bodies observed in diabetes, we find 
fever, carcinoma, inanition, lesions of the central nervous system, digestive 
disturbances, delayed chloroform poisoning, cases of pregnancy in which 
death of the fetus has occurred or in which persistent toxic vomiting is noted, 
cyclic vomiting, and other conditions associated with an increased output of the 
acetone bodies. 1 According to Mohr, most of the cases may be traceable either 
to limitation of carbohydrates in the food or to diminished power of utilizing 
them. Increased protein catabolism may play a role in the pathogenesis of this 
condition. A general statement should be that the excretion of the acetone 
bodies is little influenced by the amount of fat in the food of the normal individual, 
providing the carbohydrate content of the diet is good; but in pathologic condi- 
tions fats play a much greater role than do proteins in bringing about an 
acetonuria. 

As ^-oxybutyric acid is the mother substance from which the other acetone 
bodies are formed by oxidation, we should expect to find, as we actually do, 

1 See Fischler and Kossow, Deutsch. Arch. f. klin. Med., 1913, CXI, 479; Czapski, Arch, 
f. exper. Path. u. Pharmakol., 1914, LXXVII, 218; Zade, Arch. f. Kinderh., 1914, LXIII, 1; 
Kerley, Am. Jour. Dis. Child., 1914, VIII, 292; Peabody, Arch. Int. Med., 1914, XIV, 236; 
RoUy, Ztschr. f. klin. Med., 1914, LXXIX, 548. 



320 DIAGNOSTIC METHODS. 

the severest cases showing large amounts of ^-oxybutyric acid and small 
or even no excretion of the other members of this group. As a- rule, it may 
be said that the more acetone the less /3-oxybutyric acid but this is not always 
the case. In the diabetic coma we usually find large amounts of the first two 
members of this group while acetone may be absolutely lacking, and, on the 
other hand, we find in some of the milder types of diabetes acetone and no 
diacetic or oxybutyric acids. Dakin 1 and Wakeman and Dakin 2 have shown 
that the liver possesses two ferments by which the mutual interconversion of (3- 
oxybutyric acid and aceto-acetic acid may be effected, the one being an oxidiz- 
ing process, the other a reduction process. The acidosis may, therefore, be 
more a result of the latter than of the former action. 

(a). Acetone (CH 3 -CO-CH 3 ). 

Chemically, acetone is dimethyl ketone. It shows, therefore, the reactions 
for this group of chemical compounds but is easily confused, both with the 
aldehyds and alcohols. The urine rarely shows typical reactions for acetone 
if the older tests are applied directly to the urine, so that it is necessary to distill 
and examine the distillate. In this process diacetic acid is split up into acetone 
and carbon dioxid, so that it is impossible to tell whether acetone was preformed 
or was produced by heating. From the clinical standpoint it is a matter 
of indifference, as acetone and diacetic acid are so closely related that their 
clinical significance is the same, acetone representing merely a further stage in 
the oxidation of this group of bodies. In these tests the urine must be perfectly 
fresh. If it is desired to eliminate the influence of the diacetic acid upon the 
acetone reaction, the urine may be alkalinized with sodium hydrate and extracted 
with pure ether. The ether removes the diacetic acid salt. 
LegaPsTest (Le Nobel's Test). 

To a few c.c. of the urine are added a few drops of a fairly concentrated 
solution of sodium nitroprussid and then sodium or potassium hydrate 
until the mixture is strongly alkaline. A ruby-red color, later changing to 
yellow, appears in the presence of acetone. It will be remembered that 
this same test is given by creatinin, so that further modifications are necessary 
to permit of differentiation. If the ruby-red solution be treated with an 
excess of glacial acetic acid, the first red color will change into a carmine or 
reddish-purple color in the presence of acetone, while the same treatment 
with creatinin solutions yields a yellow, changing to green and finally to a 
blue coloration. As Le Nobel has found, ammonium hydrate does not give 
this reaction with creatinin, but with acetone, although the reaction is much 
slower in appearing. This test is given by diacetic acid, by alcohol, and by 
acetic aldehyd, so .that it is not especially distinctive for acetone. It is perhaps 
better if this test is to be used at all that the urine be previously acidified and 
distilled, the distillate yielding a reaction which is more sensitive, according 
to Studer, although diacetic acid will thus be converted into acetone. 

Lange 3 has modified this test as follows: To the suspected urine add 1/2 

1 Jour. Biol. Chem., 1910, VIII, 97. 

2 Ibid., 1910, VIII, 105. 

3 Munch, med. Wchnschr., 1906, LIII, 1764. See, also, Yanagawa, Biochem. Ztschr., 1914, 
LXI, 256. 



THE URINE. 321 

c.c. of glacial acetic acid and a few drops of a freshly prepared aqueous solution 
of sodium nitroprussid. Mix thoroughly and overlay the mixture with 2 c.c. 
of concentrated ammonium hydroxid. At the point of contact a purplish-red 
ring is observed in the presence of acetone. 

Lieben's Test. 

To a few c.c. of urine 1 or, preferably, of the distillate are added a few 
drops of concentrated sodium or potassium hydrate and a few drops of a 
solution of iodin in potassium iodid. On slightly warming the mixture yel- 
low crystals of iodoform will separate, which may be recognized by their 
characteristic odor as well as by their hexagonal shape when examined under 
the microscope. This test is given by alcohol as well as by aldehyds, and 
will show amounts of acetone varying between 1/ 100 and 1/ 1000 of a mg. 
Gunning's Test. 

This is a modification of the previous test and is much more specific, 
being given only by acetone. To the distillate from the urine are added a 
few drops of an alcoholic solution of iodin and the mixture treated with 
ammonia until a black precipitate of nitrogen iodid forms. On allowing 
the tube to stand for periods varying between 12 and 24 hours, this black 
precipitate disappears, leaving a yellow sediment of iodoform, which may be 
recognized as mentioned above. This test is less delicate than the original 
one of Lieben, detecting acetone when present in amounts of 1/100 of a mg. 
per c.c. of urine. 

Frommer's Test. 
Frommer 2 has introduced a test which seems to be distinctive for acetone 
and at the same time very delicate. It is based upon the fact that acetone re- 
acts with salicyl aldehyd to form dioxydibenzoylacetone, according to the follow- 
ing equations. The alkali salt is distinctly red. 

C 6 H 4 OHCHO + (CH 3 ) 2 CO = C 6 H 4 OHCH= CHCH 3 CO + H 2 
C 6 H 4 OHCH=CH + CHOOHC 6 H 4 =-C 6 H 4 OHCH=CH +H 2 0. 

/CO ^>co 

CH 3 CH=CHOHC 6 H 4 

The test may be performed as follows: Ten c.c. of urine are strongly 
alkalinized with potassium hydrate and 10 to 12 drops of a 10 per cent, solution 
of salicyl aldehyd in absolute alcohol are added and the mixture warmed to 
about 70 C. In the presence of acetone the fluid becomes yellow, then red, 
later purplish-red, and, on long standing, dark red. In the absence of acetone 
the color of the urine is practically unchanged. This test is said by Frommer 
to indicate the presence of one one-millionth of a gram in 8 c.c. of urine. 

Instead of applying the test as above, we may add about 1 gram of potassium 
hydrate (in the solid state) to 10 c.c. of urine and, without waiting for complete 

1 Bardach (Ztschr. f. physiol. Chem., 1908, LIV, 355) and, more recently, Rosenbloom 
(Jour. Am. Med. Assn., 1912, LIX, 445) have shown that protein and many of its derivatives 
prevent the formation of the characteristic iodoform crystals. Hence the distillate should 
always be used. 

2 Berl. klin. Wchnschr., 1905, XLII, 1008. See Engfeldt, Ibid., 1915, LII, 458. 



322 DIAGNOSTIC METHODS. 

solution to occur, treat the mixture with 10 to 12 drops of the alcoholic solution 
of salicyl aldehyd and warm to 70 . At the zone of contact of the alkali and 
the salicyl aldehyd an intense purplish-red ring is observed only in the presence 
of acetone. 

The writer has frequently used this test and finds it very satisfactory, 
as it does not react with diacetic acid unless the heating be long continued. 

Quantitative Determination of Acetone. 

Many of the quantitative methods given for acetone are more or less 
complicated and at the same time do not yield absolutely accurate results. The 
writer selects, therefore, those proving most satisfactory in his hands. All of 
these methods, with the exception of Folin's, give the amount of preformed ace- 
tone as well as that derived, by distillation, from the diacetic acid. 1 

Huppert-Messinger Method. 

The principle of this method is the determination of the amount of iodin 
necessary to transform into iodoform the acetone derived in the distillation 
of the urine. Knowing this factor, a simple calculation yields the amount of 
acetone present. 

For this determination certain solutions are necessary: 

(1). A 50 per cent, solution of acetic acid. 

(2). A tenth-normal solution of sodium thiosulphate. In preparing 
this solution 24.8 grams of crystallized sodium thiosulphate (Na^C^FLjO) 
are carefully weighed out and dissolved in distilled water, the solution being 
made in a volumetric flask and diluted exactly to the liter mark. 

(3). A tenth-normal solution of iodin. This solution requires exactly 
12.685 grams of iodin in one liter. As iodin is difficultly weighable on account 
of its volatility, it is advisable to weigh out approximately 13 grams of iodin 
and dissolve in approximately 1 liter of water to which has been added 25 
grams of potassium iodid. This solution is then standardized by titrating 
against the previously made tenth-normal thiosulphate solution, using thin 
starch paste as an indicator and adding the iodin solution from a buret until 
the blue color of iodid of starch just appears. This determination is then con- 
firmed by duplicate estimations. Twenty c.c. of the iodin solution should be 
the equivalent of 20 c.c. of the thiosulphate solution, so that the necessary 
dilution of the iodin solution may be determined by the formula previously 
given under Determination of Chlorids in the Urine (page 197). One c.c. 
of the standard tenth-normal iodin solution equals 0.012685 gram of iodin 
and represents 0.967 mg. of acetone. 

Technic. 
Five hundred c.c. of normal acid urine or 100 c.c. of acetone-rich urine 
are treated with 2 c.c. of 50 per cent, acetic acid for every 100 c.c. of urine and 
distilled until 9/10 of the volume has passed over. The distillate is collected in 
a receiving flask, which is cooled with ice and which contains water to absorb the 
acetone. The flask is tightly closed with a doubly perforated stopper, through 
one hole of which passes the tube of the condenser reaching below the surface of 
1 See Sammet, Ztschr. f. physiol. Chem., 19 13, LXXXIII, 212. 



THE URINE. 323 

the water and through the second opening a bulb filled with water to act as a 
safety bulb. The tube is then washed with distilled water and the fluid in the 
safety bulb is emptied into the receiving flask. It is important that the distilling 
flask be disconnected before the heat is shut off, as otherwise the fluid might suck 
back. This distillate is treated with calcium carbonate to remove any nitrous or 
formic acid which may have distilled over, and the mixture thoroughly shaken. 

This distillate is then acidified by the addition of 1 c. c. of dilute sulphuric 
acid (diluted eight times) and redistilled until one-tenth of the volume remains 
in the distilling flask. This second distillate is received in a flask arranged 
as in the previous distillation. It is then poured into a flask which can be closed 
with a tight-fitting glass stopper. Distillate and wash-water must not fill 
the vessel more than one-third full. A large excess of carefully measured 
N/10 iodin solution is added, the mixture well shaken, and strong sodium 
hydrate solution added drop by drop. The flask is then stoppered, shaken 
for one-fourth minute, and allowed to stand for five minutes. The stopper is 
then removed, the fluid clinging to it washed into the flask, and the fluid acidified 
with strong HC1. The excess of iodin is then determined by allowing N/10 
thiosulphate solution to flow from a buret until the mixture is but slightly 
yellow, when a few c.c. of starch paste are added and the titration continued 
until the blue color just disappears. One c.c. of iodin solution used by the 
fluid corresponds to 0.967 mg. acetone, so that all that is necessary is to subtract 
the number of c.c. of thiosulphate used from the number of c.c. of iodine solution 
added and multiply by the above factor. The result is the acetone in the amount 
of urine taken. 

This method yields results which are from 5 to 10 per cent, low, 1 so that 
slight variations in the acetone excretion, as determined by this method, have 
little significance. 

Folin's Method. 

This method 2 yields only the preformed acetone occurring in the urine 
and does not regard the acetone which may be derived from the diacetic acid. 
If the urine be distilled and this method employed, the total possible acetone 
will result. It is somewhat more accurate than the previous method and is 
much more simple and less time-consuming. The writer finds it extremely 
serviceable and adopts it in all acetone determinations. 

Twenty-five c.c. of urine are measured into an aerometer cylinder, similar 
to that used in Folin's ammonia apparatus, a few drops of 10 per cent, phosphoric 
acid, 10 grams of sodium chlorid, and a little petroleum are added. In the 
absorbing bottle which is fitted with an absorption bulb are placed 300 c.c. of 
water, 20 c.c. of 40 per cent, potassium hydrate, and an excess of tenth-normal 
iodin solution. The apparatus is connected with the filter-pump in the same 
manner as described under Ammonia and an air-current drawn through for 
one-half hour. The air should not be passed as rapidly as in the ammonia 
determination. Each worker should check his air blast by control estimations, 
using known solutions of acetone. In this way the acetone will be removed 

1 See Marriott, Jour. Biol. Chem., 19 13, XVI, 281. 

2 Jour. Biol. Chem., 1907, III, 177; also, Folin and Denis, Ibid., 1914, XVIII, 263. 



324 DIAGNOSTIC METHODS. 

from the urine and converted, in the receiving flask, into iodoform. The contents 
of the receiving flask are acidified with concentrated HC1, using 10 c.c. of acid 
for every 10 c.c. of alkali previously used, and the excess of iodin titrated 
with tenth-normal sodium thiosulphate as in the previous method. 

If this method be employed with the distilled urine, subtract from the 
total acetone, obtained in the distillate, the preformed acetone, .derived from the 
fresh urine, and obtain the acetone referable to diacetic acid. If this value be 
multiplied by 1.758 the result will be the amount of diacetic acid, as such, 
present in the urine taken. 

Shaffer has recently introduced a method for the determination of the 
total acetone, which is closly related to his method for estimating ^-oxybutyric 
acid and will be discussed in a later section. 

(b). Diacetic acid (CH 3 -CO-CH 2 -COOH). 

This substance is derived from /?-oxybutyric acid and is the precursor 
of acetone. As a rule, if acetone be present in large amounts, diacetic acid 
is also present and indicates a much graver condition than does the mere 
presence of acetone. If Folin's contentions are true, most of what we now 
call acetone is, in realty, diacetic acid, so that this latter substance should be 
tested for in routine urinary work. The remarks previously made regarding 
the appearance of acetone in the urine also hold for diacetic acid. 1 This 
substance is very volatile and disappears from the urine in a relatively short 
time so that the tests should be applied to perfectly fresh specimens. 
Gerhardt's Test. 

To 10 c.c. of urine are added a few drops of 10 per cent, ferric chlorid 
solution. This is best added as long as a precipitate of phosphates occurs, 
these latter bodies being then filtered off. To the filtrate are added a few 
drops more of ferric chlorid solution when the urine shows a Bordeaux-red 
color in the presence of diacetic acid. This color appears cherry-red by 
transmitted light and purplish-red by reflected light. 

Unfortunately, however, this test is not specific for diacetic acid. 2 A 
red color is observed in the presence of cyanates, formates, acetates, phenol 
compounds, salicylates, the conjugated glycuronates of phenacetin, antipyrin, 
thallin, kryofin, and kairin, as well as meconic acid which may be excreted 
after intake of opium. If the urine be heated, diacetic acid decomposes more 
or less completely into acetone and carbon dioxid, so that the red color due to 
diacetic acid will either disappear or become much weaker, while that with the 
other substances mentioned is not affected by heat. This same disappearance 
of color is noted if the mixture be allowed to stand from 24 to 48 hours. Schrei- 
ber recommends filtration of the urine through animal charcoal, using about 10 
grams of charcoal to 100 c.c. of urine. Antipyrin, phenacetin, and kryofin 
are retained while sufficient of the diacetic acid passes through to permit of 
detection. The urine may even be acidified with sulphuric acid and extracted 
with ether. This ethereal solution, which contains the diacetic acid, is then 

1 See Dakin and Dudley, Jour. Biol. Chem., 19 13, XVI, 515. 

2 See Steensma and Koopman, Nederl. Tijdschr. v. Geneesk., 1914, LVIII, 800; Lichtwitz, 
Berl. klin. Wchnschr., 191 5, LII, 399. 



THE URINE. 325 

shaken with water and ferric chlorid solution added. A deep red color will be 
seen in the watery layer in the presence of diacetic acid. If the red color, on 
addition of ferric chlorid, be due to the presence of meconic acid, it will disappear 
on the further addition of stannous chlorid or of alkali hypochlorites, while 
that due to diacetic acid is unaffected. This test is, perhaps, more frequently 
used than any other test for diacetic acid in the urine, but is by no means as 
serviceable as the following. 
Arnold's Test. 

For the performance of this test two reagents are employed: (1) a solution 
consisting of 1 gram of paraamidoacetophenon, 100 c.c. of distilled water, 
and 2 c.c. of concentrated hydrochloric acid; (2) 1 per cent, sodium nitrite 
solution. Fifteen c.c. of urine are treated with a mixture consisting of 10 c.c. of 
solution 1 and 5 c.c. of solution 2 and one drop of concentrated ammonia added. 
In practically all urine, whether it contains diacetic acid or not, a brownish-red 
coloration is observed which changes in the absence of diacetic acid into yellow 
if the mixture be treated with an excess of concentrated hydrochloric acid, 
while if diacetic acid be present the color changes to a beautiful purple on the 
addition of acid. If the mixture be shaken the foam also shows a distinct 
violet coloration. 

As this test is somewhat difficult in the presence of small amounts of 
diacetic acid, Lipliawsky 1 has modified it as follows: Six c.c. of solution 1 
and 3 c.c. of solution 2 are treated with the same volume of urine, a drop 
of ammonia is added and the mixture shaken, when it assumes a brick-red 
color. According to the probable contents of the urine in diacetic acid 10 
drops to 2 c.c. of this mixture are treated with 15 to 20 c.c. of concentrated 
hydrochloric acid, 3 c.c. of chloroform and two to four drops of ferric chlorid 
solution. The test-tube is then closed with a cork and gently shaken for one- 
half to one minute. In the presence of traces of diacetic acid the chloroform 
assumes a characteristic violet coloration, while in the absence of this acid the 
color is yellow or light red. This test is positive for one part in 400,000 of 
water. Acetone and /?-oxybutyric acid do not react with this test nor do the 
drugs previously mentioned under the Gerhardt Test interfere. If the urine 
is highly colored it is advisable to filter through animal charcoal. 

(c). /?-Oxybutyric Acid 2 (CH 3 -CHOH-CH 2 -COOH). 

This acid, the mother substance of the acetone bodies, is found in the 
urine in extreme cases of the conditions described under Acetone. The amount 
excreted may vary from traces to as high as 100 grams. Kulz has reported 
an excretion of 225 grams in 24 hours. The free acid is practically never 
found in the urine, being excreted either as the ammonium, sodium, or 
potassium salt. These salts as well as the free acid are levorotatory and may 
be detected by the polariscope after previous fermentation of the carbohydrates. 

In the condition known as diabetic coma a specific intoxication with 
^-oxybutyric acid is assumed as the causative factor. While this is undoubtedly 

1 Deutsch. med. Wchnschr., 1901, XXVII, 151. 

2 The more scientific name for this body is &-hydroxybutyric acid. See Sassa, Biochem. 
Ztschr., 1914, LIX, 362; Kennaway, Biochem. Jour., 1914, VIII, 230 and 355. 



326 DIAGNOSTIC METHODS. 

true to a large extent it cannot be regarded as the only factor in diabetic coma, 
as administration of ^-oxybutyric acid in large quantities will not necessarily 
lead to such a syndrome unless largely retained. We do undoubtedly have an 
acidosis which acts by ultimately depriving the tissues of fixed alkalies and 
may be regarded, therefore, as of great importance in following a diabetic case. 
Were this acid, per se, accountable for the entire symptomatology, we should 
be able, by administering alkalies, to overcome the effect of the acid intoxication. 
In some cases such therapy is extremely beneficial, while in others it is practi- 
cally useless, as it cannot influence the formation of the toxic bodies and may 
not increase their elimination. 

As previously stated, the ammonia output of the urine is an invaluable 
guide in following the course of an acidosis, especially in diabetes. One 
gram of ammonia (NH 3 ) is equivalent to 6.12 grams of /?-oxybutyric acid, 
especially when in excess of the amount directly due to the food. 

To detect /?-oxybutyric acid follow the method of Hart. 1 To 20 c.c. of 
urine add 20 c.c. of water and a few drops of acetic acid and boil until the 
volume is reduced to about 10 c.c. (to remove acetone and diacetic acid). To 
the residue add sufficient water to make the volume 20 c.c. and place 10 c.c. in 
each of two test-tubes. To one of these add 1 c.c. of hydrogen peroxid, warm 
gently and allow to cool. Apply Lange's test for acetone (see p. 320) and allow 
the tubes to stand for a few hours. No reaction is observed in the tube which 
contains no hydrogen peroxid, while a distinct red zone is seen in the other. 

Quantitative Determination. 

A large number of methods have been advanced for the determination 
of /3-oxybutyric acid in the urine. Among these we find the methods of Kiilz, 
Tollens, Wolpe, Magnus-Levy, Bergell, Stadelmann, and Darmstadter. While 
some of these are accurate under certain conditions, so many precautions 
must be taken that widely varying results may be obtained. On the other hand, 
the method of Magnus-Levy, which is undoubtedly very exact, requires 24 hours, 
while that of Bergell depends to a large extent upon the condition of the powder 
which is extracted with ether. 

Blacks Method. 
This method 2 is a modification of those of Magnus-Levy and of Bergell, 
but in the writer's hands has given much more satisfactory results. One 
hundred c.c. of urine are faintly alkalinized with sodium carbonate and evapo- 
rated in a porcelain dish to one-third or one-fourth of the original volume. The 
residue is then concentrated to about 10 c.c. on a water bath in order completely 
to remove the diacetic acid. This is then cooled, acidified with a few drops 
of hydrochloric acid, and made into a thick paste with plaster of Paris. , This 
mixture soon begins to set, when it is stirred and broken up with a glass rod. 
This porous mass is then transferred to a Soxhlet apparatus and extracted 
with pure ether for two hours. The ether extract is evaporated, the residue 
taken up with water, decolorized with bone-black and filtered perfectly clear. 

1 Am. Jour. Med. Sc, iqoq, CXXXVII, 869. 

2 Jour. Biol. Chem., 1908, V, 207. 



THE URINE. 327 

The filtrate is then made up to 25 c.c. and its rotation determined with a 
polariscope. In determining the amount of /?-oxybutyric acid from its rotation 
we must make use of the following calculation. The specific rotation of the 
free acid is — 24.12 in a decimeter tube. One division of the scale in the case of 
glucose equals 2 per cent., so that we may find the percentage of /3-oxybutyric 
acid by the following proportion : 

2 : X : : 24.12 : 52.7. X = 4.37 per cent. 

By a similar proportion it may be found that one division of the scale corre- 
sponds to 7.1 per cent, of sodium /?-oxybutyrate, whose specific rotation 

is —I4-35- 

This method is just as exact as those of Magnus-Levy and Bergell and 
has the advantages that it is simpler, more reliable, and may be performed 
in shorter time. The greatest difficulty with this method arises in the deter- 
mination of the exact point of the scale at which the two portions of the polari- 
metric field are equally illuminated. Magnus-Levy has shown that a difference 
of 1/10 of i° in reading the polariscope amounts to about three grams per 
liter of /?-oxybutyric acid, so that great care must be used with the polariscope 
in this method as well as in all others in which it is applied. 

Shaffer's Method. 

This method 1 seems to the writer to be the most desirable one that has 
been advanced. Shaffer and Marriott 2 show that this method yields uniformly 
about 90 per cent, of theoretical values. The results obtained must, therefore, 
be corrected by the addition of 10 per cent, of the amount found. See, also, 
Pribram 3 in this connection. 

The principle of the method is the oxidation of /?-oxybutyric acid to acetone 
and carbon dioxid and the determination of the amount of acetone thus evolved. 
The acetone and diacetic acid already existing as such in the urine are previously 
determined by this method, so that it serves as a general one for the acetone bodies. 

From 25 to 250 c.c. of urine, depending upon the amount of ^-oxybutyric 
acid expected, are measured into a 500 c.c. volumetric flask and an excess of 
basic lead acetate and 10 c.c. of concentrated ammonia are added. In selecting 
the amount of urine to be taken one should use sufficient to yield from 25 to 
50 mg. of acetone derived from /3-oxybutyric acid. The solution in the flask 
is then diluted to the graduating mark, is thoroughly shaken, and filtered. 
Two hundred c.c. of the filtrate (representing 2/5 of the original volume of 
urine taken) are diluted with water to 500 or 600 c.c. 15 c.c. of concentrated 
sulphuric acid and a few grams of talcum are added, and the mixture distilled 
until 200 to 250 c.c. of distillate collects (distillate A). 

In this distillation, the distilling flask, which may be an 800 c.c. Kjeldahl 
flask, should be fitted with a dropping funnel and water run in to prevent 
the volume of fluid in the flask from becoming less than 400 c.c. This distillate 
(A) contains the preformed acetone and that from the diacetic acid as well 

1 Jour. Biol. Chem., 1908, V, 211. 

2 Jour. Biol. Chem., 1913, XVI, 265. 

3 Ztschr. f. exp. Path. u. Therap., 1912, X, 279 and 284. 



328 DIAGNOSTIC METHODS. 

as volatile fatty acids which may be present in the urine. To remove the fatty 
acids, especially formic acid, the distillate A is redistilled after adding 5 c.c. 
of 10 per cent, sodium hydrate solution. This distillate (A 2 ) is then titrated 
with standard tenth-normal iodin and thiosulphate solutions as described 
in the Huppert-Messinger method for determination of acetone. 

The residue of urine and sulphuric acid from which A was obtained is 
again distilled, dropping in 400 to 600 c.c. of 0.1 per cent, to 0.5 per cent, potas- 
sium bichromate 1 solution. This bichromate must not be added faster than 
the distillate collects unless the boiling liquid turns a pure green color, indicating 
that the bichromate is being used up more rapidly. When about 500 c.c. of 
distillate (B) have collected, 20 c.c. of 3 per cent. H 2 2 are added to the distil- 
late together with a few c.c. of sodium hydrate solution and this redistilled. 
This second distillate (B 2 ) is then titrated with tenth-normal iodin and thio- 
sulphate solution. One mg. of acetone represents 1 .794 mg. of /3-oxy butyric acid. 

(4). Abnormal Pigments. 
(a). Blood Pigments. 

The principal blood pigment appearing in the urine is hemoglobin, which 
has been previously discussed under the heading of Protein in the Urine. Cer- 
tain derivatives of this pigment are found, however, in conditions in which 
hemoglobin does not appear (see hemoglobinuria). 

Hematoporphyrin . 

This is an iron-free derivative of hemoglobin and appears to be present 
in minute traces in normal urine. Pathologically, it has been found in cases 
of rheumatism, phthisis, Addison's disease, pericarditis, paroxysmal hemoglo- 
binuria, cirrhosis of the liver, exophthalmic goiter, croupous pneumonia, 
lead poisoning, syphilis, and many acute infectious diseases. Long-continued 
use of certain hypnotics, such as sulphonal, 2 trional, and tetronal, is fre- 
quently associated with the appearance of hematoporphyrinuria. 

Urine containing hematoporphyrin is usually dark-red in color, but may 
vary from a brownish-red or port- wine color to a distinct Bordeaux-red. Ham- 
marsten has shown that this color is not entirely due to the hematoporphyrin, 
but partially to other abnormal pigments whose identity is not certain. 

In examining urine for the presence of hematoporphyrin the spectro- 
scopic method is practically the only one available. If this pigment be present 
in large amounts the urine may be directly examined with the spectroscope, 
showing the four bands of alkali hematoporphyrin discussed in the section 
on Blood. As this method is not always certain and not always easy of applica- 
tion, it would seem preferable to treat 50 c.c. of urine with 10 c.c. of 10 per 
cent, sodium hydrate solution. The precipitated phosphates carry down 

1 Cooke and Gorslin (Jour. Biol. Chem., 1911, X, 291) have wisely advised the following 
modification. The residue of urine and sulphuric acid is diluted to 600 c.c. and 5 c.c. of a 5 
per cent, solution of potassium bichromate are added. Distill down to 300 c.c. and add 
water through the dropping tube to 500 c.c. Distill until 500 c.c. have been obtained. Ad- 
ditional 5 per cent, bichromate is run in, a few cubic centimeters at a time, whenever the 
distilling solution shows a greenish tinge. 

2 See Pfortner, Deutsch. med. Wchnschr., 1914, XL, 1563; Fischer, Ztschr. f. plrysiol. 
Chem., 1915, XCV, 34. 



THE URINE. 329 

the pigments. This precipitate is then treated with 10 drops of concentrated 
hydrochloric acid and 15 c.c. of absolute alcohol. The solution is filtered 
if necessary and examined with the spectroscope when the two absorption 
bands of acid hematoporphyrin will be observed. 

(b). Biliary Pigments. 

Normally, bile pigments do not occur in the human urine. As was pre- 
viously discussed in the section on Urobilin, biliary pigments may appear 
in the urine in conditions interfering with the passage of the bile into the 
intestine or when increased formation of biliary pigments from blood pigments 
has occurred and an associated obstruction of the bile-ducts is present. 

We see, therefore, that choluria occurs in every case in which there is 
obstruction to the outflow of bile into the intestine. Thus in catarrhal jaundice, 
biliary calculi in the common duct, carcinoma of the liver, and cirrhosis, 
bilirubinuria is frequent. Moreover, we may also find biliary pigments arising 
from purely hematogenous conditions, such as pernicious anemia, malaria, 
typhoid fever, arsenical poisoning, and yellow fever. Whether this latter 
type is not really hepatogenous in origin, as Stadelmann believes, is still un- 
settled, but it would seem more plausible to assume a primary breaking down 
of the red cells and a secondary insufficiency of the liver. 

The chief biliary pigment found in the urine is bilirubin, 1 which is inter- 
mediate between hemoglobin and urobilin. On oxidation of this pigment, 
either in the system or in the methods of examination, various other pigments 
may arise. Thus we may find biliverdin, bilicyanin, bilifuscin, biliprasin, 
cholecyanin, and choletelin. In the fresh urine bilirubin is the only pigment 
noted. 

A bile-containing urine may show various shadings of color, ranging 
from a greenish-yellow, through yellowish-brown, deep brown, or greenish- 
brown, to a pure green. If the urine be shaken, a yellowish or greenish-yellow 
foam is observed, while in normal urines the foam is practically colorless. 
The presence of an excess of urobilin may also give a brownish foam. Urine 
containing bile always shows the presence of nucleoalbumin along with 
slight traces of serum albumin, so that bile should be regarded as a source 
of extraneous albumin. The sediment will usually be more or less colored 
by the biliary pigment, the casts in yellow fever, for instance, being usually 
distinctly bile-stained. 

Qualitative Tests. 

A large number of tests have been advanced for the detection of biliary 
pigments in the urine and many of them are distinctly unsatisfactory. If 
large amounts of bile be present, the urine, acidified with HC1, may be shaken 
out with chloroform which dissolves the bilirubin. If the chloroform be 
evaporated, rhombic crystals with rounded edges or distinct needles of a 
brownish-red color will be observed. These crystals will give the color tests 
mentioned below. 

1 See van den Bergh and Snapper, Nederl. Tijdschr. v. Geneesk., 191 5, II, 469, Kiister, 
Ztschr. f. physiol. Chem., 191 5, XCIV, 136. 



330 DIAGNOSTIC METHODS. 

Smith's Test. 
This test has been described under other names, as those of Trousseau, 
Kathrein, Rosin, and Marechalt. A few c.c. of urine, acidified if necessary 
with acetic acid, are treated with a i per cent, alcoholic solution of iodin in 
such a way that the latter solution is superimposed upon the urine, forming 
a distinct line of contact. If bilirubin or other biliary pigments be present, 
a beautiful emerald-green color is observed at the point of contact. This test 
is especially recommended, but it is not very sensitive, indicating only one part 
of biliary pigment in 10,000 of urine. Certain drugs, especially antipyrin, 
may lead to the formation of a green color with this test. Thymol, if used 
as a preservative, may give rise to confusion with this and other bile tests. 

Gmelin's Test. 

One or two c.c. of nitric acid are placed in a test-tube and the same amount 
of urine is allowed to flow from a pipet in such a way that a distinct contact 
line is formed. If the nitric acid contains a trace of nitrous acid and the 
urine biliary pigment, a distinct green ring will be observed at the line of 
contact. In some cases, as the nitric acid oxidizes the pigment, a play of 
colors may be seen from green, through a blue, violet, and red, to a yellow. 
The primary green is the characteristic color, other colorations being occasion- 
ally due to pigments other than biliary. This test is supposed to indicate 1 
part of bilirubin in 80,000 of urine. 

If the urine contains an excess of indican a deep-blue ring may be observed 
or the combination of this blue with the yellow of the urine may give a green. 
In the presence of skatoxyl a violet-red ring may be noted, while various 
medicaments may give colorations ranging through the entire spectrum. In 
such cases it is always advisable to extract the acidified urine with chloroform 
and apply the test either to the evaporated residue or to its aqueous solution. 

This test is, perhaps, more frequently used than any of the biliary tests, 
but requires considerable experience for its proper interpretation. If the 
urine be diluted the test is somewhat more distinctive. Many modifications 
of this test have been advocated, the most serviceable being the following: 

Rosenbach's Test. 
A large quantity of urine, which has been acidified with HC1, is filtered 
several times through a thick filter-paper, which will hold back the bile-stained 
elements of the urine. It is sometimes advisable to add a little milk of lime 
to the urine before filtering, instead of the HC1, as this will throw down the 
phosphates which will carry with them the biliary pigment. If the filter- 
paper and contents be dried by pressing with a second dry filter-paper and 
a drop of yellow nitric acid allowed to fall upon it, distinct rings will be seen, 
which will be colored as in the previous test, the green one being external. 

Nakayama's Test. 
This is a modification of the older Huppert test. 1 Five c.c. of acid urine 
are treated with an equal volume of 10 per cent, barium chlorid solution 
and the mixture centrifuged. The barium chlorid precipitates the phos- 

1 See Rosenbloom, New York Med. Jour., 1914, XCIX, 229. 



THE URINE. 331 

phates and sulphates and carries down the biliary pigments. The superna- 
tant fluid is then poured off and 2 c.c. of the following reagent are added to 
the precipitate. The reagent consists of 99 c.c. of 95 per cent, alcohol, 1 c.c. 
of concentrated HC1, and 0.4 gram of ferric chlorid. If this mixture of 
precipitate and reagent be heated to boiling, a bluish-green or a brilliant green 
solution is obtained, which becomes violet or red on the addition of nitric acid. 
This test is said to indicate one part of bilirubin in 1,200,000 parts of urine. 

Hammarsten's Test. 

As the reagent in this test we use a mixture of one part of 25 per cent.HN0 3 
and 19 parts of 25 per cent. HC1. Beforeuse one part of this reagent is mixed 
with four parts of alcohol. A few drops of the urine are added to this mixture, 
when it assumes a green color in the presence of biliary pigments. If the 
urine be treated as in Nakayama's test and the precipitate mixed with 1 to 2 c.c. 
of the acid alcohol reagent and the whole centrif uged for a short time, a green 
solution is obtained if one part of bilirubin in 1,000,000 of urine be present. 

Bile Acids. 

The bile acids, taurocholic and glycocholic acids, are found in the 
urine in the form of their sodium salts. They may be found in small amounts 
in the same conditions in which the biliary pigments are present, but their 
amount is usually so small that they cannot be detected, as a rule, without 
being previously isolated. It seems to be fairly well established that the 
bile acids must be present to the extent of 1/2 per cent, before detection in the 
urine is possible. 

As the clinical significance of these acids is the same as that of the pig- 
ment and as their amount is so small that the methods of isolation require 
a fairly large volume of urine, the writer will refer to works on physiologic 
chemistry for such procedures. 

No absolutely reliable test is known for the detection of the bile acids 
in the untreated urine. There are, however, a few tests which are occasionally 
given providing the bile acids be present in sufficient amount. 

Hay's Test. 

This test depends upon the reduction of the surface tension of the urine 
in the presence of the bile acids. As advocated by Beddard and Pembrey, 
a pinch of powdered sulphur is sprinkled upon the surface of urine which 
should be preferably at a temperature not over 17 C. In normal urines the 
sulphur will float upon the surface, while if the urine contains bile acids the 
sulphur may sink at once indicating one part in 10,000 or may sink only after 
a few seconds to one minute, in this latter case indicating one part in 50,000. 
According to Sahli this test does not discriminate between biliary acids and 
biliary pigments, but clinically it is a matter of indifference which one is present. 
Phenol or aniline compounds lower the surface tension of the urine so that their 
presence may lead to wrong conclusions. 1 

1 See Allen (Jour. Biol. Chem., 1915, XXII, 505) for a method of measuring accurately the 
variation in surface tension of the urine. 



332 DIAGNOSTIC METHODS. 

Oliver's Test. 
This test is based upon the well-known property possessed by the bile 
acids of precipitating peptone when in acid solution. The reagent is as follows: 

Powdered peptone, 8.33 grams 

Salicylic acid, 1.12 grams 
Acetic acid, 2 drops 

Distilled water, 1 liter 

One to two c.c. of clear filtered urine are placed in a test-tube and treated 
with 5 c.c. of the reagent. In the presence of bile acids a decided milkiness 
appears at once, being the more intense the larger the amount of bile acids. 

Other tests, such as those of Pettenkofer and Udranzky, are only ser- 
viceable in the testing of the isolated bile acids. 

(c). Melanin. 

In cases of melanotic tumors the urine not infrequently contains a chro- 
mogen (melanogen), which is converted into melanin on allowing the urine 
to stand or after adding alkalies or oxidizing agents. The urine when freshly 
voided is normal in color, but on exposure to air gradually darkens until it 
becomes distinctly black. This coloration is first noticed in the upper portion 
of the urine and gradually extends toward the bottom. If ferric chlorid 
be added to the urine the coloration may be somewhat intensified so that one 
may assume an excess of phenol derivatives. It is to be remembered that 
indican must first be split up with acid before giving the coloration with ferric 
chlorid; moreover, melanin is insoluble in chloroform while the indigo is 
readily soluble. (See Eppinger. 1 ) 

The addition of the ferric chlorid may produce a black precipitate which 
is soluble in sodium carbonate solution, from which it may be reprecipitated 
by mineral acids. This pigment may also be found in some cases of chronic 
malaria 2 so that it is not absolutely pathognomonic of melanotic tumors. 

(d). Phenol Derivatives. 

As previously mentioned in the discussion of the variations in color of 
the urine, it may be dark colored either on voiding or after standing. While 
such colorations may be due to melanin, they are much more frequently trace- 
able to the presence of sulphuric acid in conjugation with phenol, 3 paracresol, 
pyrocatechin, and hydroquinone. These substances are excreted especially 
in conditions associated either with increased intestinal putrefaction or with 
putrefactive processes elsewhere in the system. Aside from direct poisoning 
with these substances, they may be regarded as having much the same clinical 
significance as indican. 

Qualitative and quantitative tests for these substances are rarely of clin- 

1 Biochem. Ztschr., 1910, XXVIII, 181. 

2 Urriola (Interstate Med. Jour., 1912, XIX, 74) claims that a urinary excretion of blood 
pigment (probably hematin and not melanin) is invariably present as a pathognomonic sign of 
malaria. This may appear in the centrifuged specimen as a very abundant intense black pig- 
ment; a blue pigment, very constant but small in amount; or as an ochre pigment occasionally. 

3 See Hensel, Ztschr. f. physiol. Chem., 1912, LXXVIII, 373; also, Folin and Denis, 
Jour. Biol. Chem., 1915, XXII, 305 and 309; Siegfried and Zimmermann, Biochem. Ztschr. 
1915, LXX, 124. 



THE URINE. 333 

ical importance. They may be roughly determined by estimating the amount 
of ethereal sulphates in the urine, when a large increase may be assumed 
to be referable to these bodies unless indican is greatly in excess. 

(e). Alkapton. 

In certain conditions of disturbed protein metabolism, the urine contains 
pigments which cause it to turn dark on the addition of alkali or on standing. 
These urines are normal in color when voided and become black almost immedi- 
ately on the addition of alkali, whence the name alkapton bodies and alkaptonuria 
for their excretion in the urine. The substances causing this change of color 
are hydroquinon-acetic acid (as it has been prepared synthetically from 
gentisic aldehyd, it has been called homogentisic acid with the formula 
C6H 3 (OH) 2 (CH2COOH)) and uroleucic acid, whose structure has not been 
absolutely settled. 1 The former of these acids is present in all cases of alkap- 
tonuria, while the latter may be present in all cases, but in such small amounts 
that it remains undetected. Urines containing these bodies strongly reduce 
copper and ammoniacal silver solutions, while bismuth solutions are little 
affected. The so-called glycosuric acid of Marshall is probably identical 
with homogentisic acid and is not a definite chemical entity. 

This condition is observed at various periods of life and seems to be of 
theoretical more than practical interest, as of the 48 cases reported none ap- 
peared to be much disturbed in health, the condition usually being detected 
accidentally. 2 

The source of these substances is still uncertain. Baumann and Wolkow 
believe that the influence of specific bacteria in the intestinal canal upon 
the tyrosin, formed in the hydrolytic cleavage of the proteins, leads to direct 
formation of homogentisic acid. While this accounts for some of the ab- 
normal substance, it cannot account for the entire amount of 3 to 7 grams of 
homogentisic acid excreted in the 24 hours. From the work of Meyer, Falta, 
Langstein and Wohlgemuth it would appear that this condition depends not 
on an abnormal formation of homogentisic acid but on an incapacity for further 
oxidizing it when formed. Dakin 3 believes that both factors play a part. 
However this may be, alkaptonuria has very few ill-effects, although transitory 
symptoms have been observed in occasional cases of diabetes, cirrhosis of the 
liver, tuberculosis, pyonephrosis and gastritis. The browning of the cartilage 
occurring in ochronosis is supposed by Albrecht and Zdarek to have some rela- 
tion to the alkapton bodies, although Langstein could not show the alkapton 
acids in the urine in such cases. 

Qualitative and quantitative tests for these acids are rarely necessary. 
The characteristic change of color on alkalinizing the urine is, at least, sug- 
gestive of homogentisic acid. Like all other hydroxy derivatives of benzol, this 
substance shows the Schiff reaction with ferric chlorid, producing a transitory 

1 See Mdrner, Ztschr. f. physiol. Chem., 1912, LXXVIII, 306; Oswald (Ztschr. f. physiol. 
Chem., 1 914, XCIII, 307) believes this acid is identical with homogentisic acid. See, also, 
Gross, Biochem. Ztschr., 1914, LXI, 165. 

2 See Poulsen, Munch, med. Wchnschr., 1912, LIX, 364; also, Baldwin, Am. Jour. Med. 
Sc, 1913, CXLV, 123. 

3 Jour. Biol. Chem., 1911, IX, 151. See, also, Pincussohn, Ergeb. d. inn. Med., u. d. Kinder- 
heilkde., 1912, VII, 454; Soderbergh, Nord. Med. Arch., 1915, XLVIII, 1. 



334 DIAGNOSTIC METHODS. 

blue coloration when present in amounts of i to 4,000. The reduction of 
copper solutions should not be mistaken for sugar, as homogentisic acid neither 
ferments nor shows any optical activity. The urine will usually show a relatively 
high acidity. As this substance is practically harmless in the system we should 
expect to find, as we actually do, no increase in the conjugated sulphates or 
glycuronates nor any increase in the ammonia content of the urine (Neuberg). 

(/). Ehrlich's Diazo Reaction. 

Under certain pathologic conditions the urine has been found to contain 
a chromogen which gives a deep red color to the urine when treated with 
diazo compounds and ammonia. This is known as the diazo reaction, the 
urinary substance causing it being somewhat uncertain. According to Bond- 
zynski, the alloxyproteic acid is the causative factor, but this needs confirma- 
tion, as Clemens has apparently shown that the body producing the reaction 
is sulphur-free. 

In the performance of this test two reagents are used: (1) sulphanilic 
acid 5 grams, 50 c.c. of concentrated hydrochloric acid, and 1,000 c.c. of 
water; (2) 1/2 per cent, aqueous sodium nitrite solution. 

To a mixture consisting of 50 parts of solution 1 and one part of solution 
2 is added an equal volume of the urine. The mixture is then shaken and 
about one-tenth of the bulk of ammonia added quickly, and the mixture thor- 
oughly shaken. The ammonia may be added in such a way that a line of 
contact forms, this latter method frequently bringing out a much more beauti- 
ful reaction. According to Greene, a mixture consisting of 100 parts of solution 
1 to one of solution 2 renders the test more delicate. Instead of sulphanilic 
acid, paraamidoacetophenon may be used, as suggested by Friedenwald. 

If the test be positive the entire urine will assume an intense red coloration 
or a colored ring will be observed at the point of contact between the ammonia 
and the mixture. With normal urine a distinct orange color may be observed. 
On shaking the mixture the foam will be more or less brilliant red in color, 
which is more characteristic than the red coloration of the mixture. On al- 
lowing the mixture to stand for 24 hours a green precipitate may be observed 
at the bottom of the tube, which Ehrlich regards as especially characteristic 
of the true diazo reaction. This green precipitate is not always present and 
is not necessary for a positive reaction, the red coloration of the mixture and 
of the foam being more frequent. 

The administration of certain drugs may markedly affect this test. 1 Thus 
we find that naphthalin, chrysarobin, opium, and phenol derivatives give 
a reaction which is very similar to the true diazo reaction, but may be distin- 
guished, according to Wood, by the fact that color is more permanent in alka- 
line solutions, does not fade to any extent on the addition of a strong mineral 
acid, the foam is more yellow than in the typhoid reaction, and the green pre- 
cipitate does not appear on standing. Burghart has found that, following 
the administration of tannic acid, gallic acid, tannigen, and tannalbin, the 
diazo reaction disappears from the urine, the inhibiting effect being perhaps 

1 Skorczewski und Sohn (Wien. klin. Wchnschr., 19 11, XXIV, 1700) show that the urine 
of patients taking atophan gives this reaction. 



THE URINE. 335 

exerted upon the reagents used rather than upon the unknown factor which 
usually causes the coloration. 

According to Ehrlich, if the urine contains an excess of biliary pigment, 
a dark cloudy discoloration may occur, which is changed on boiling to a dis- 
tinct reddish violet. He also finds on applying the diazo test in some cases 
that the urine and foam become yellow before the addition of ammonia. After 
ammonia is added the color changes to a lighter yellow. This reaction is known 
as Ehrlich's "egg-yellow" reaction, and is supposed to be due to the presence 
of urobilinogen. He regards this latter test as especially important in pre- 
dicting the crisis of pneumonia. 

This diazo reaction is never positive in health. It was formerly regarded 
as pathognomonic of typhoid fever, but it has been shown to occur in many 
other conditions. It is true that in typhoid fever it may be present as early 
as the third or fourth day and may persist for some time, reappearing if a 
relapse occurs, while the Widal test, as will be remembered, does not vary 
with a relapse. Moreover, the intensity of this reaction is somewhat parallel 
with the severity of the case, while the Widal reaction may not be present 
in the severest types of typhoid fever. A positive diazo reaction occurs fre- 
quently in measles, somewhat less frequently in pneumonia, miliary tubercu- 
losis, scarlet fever, 1 diphtheria, and erysipelas, while in rheumatism and menin- 
gitis it is rarely obtained. Michaelis believes that the presence of a positive 
diazo reaction in pulmonary tuberculosis indicates a progressive condition 
with a grave prognosis. 2 

This reaction is, therefore, a valuable aid in diagnostic work. As it 
is present in about 80 per cent, of cases of typhoid fever, a negative reaction 
would not necessarily exclude typhoid nor would a positive reaction prove 
its presence. Its appearance in a relapse is of value, as the Widal test would 
give no information under such conditions. It is to be said, however, that 
the diagnosis should by no means rest upon this test. 

(g). Russo's Test. 

This test 3 has been recently advanced and seems to have somewhat 
more diagnostic importance than has the diazo reaction. The technic 
is very simple and is as follows: Four drops of a 1 to 1,000 aqueous solution 
of methylene blue are added to 4 or 5 c.c. of suspected urine. If the reaction 
be positive the mixture turns to an emerald or mint-green hue. A light green 
or bluish-green tint shows a negative reaction. The positive reaction is not 
affected by boiling the urine or by the previous ingestion of such compounds 
as phenacetin, salol, quinin, and calomel. The difficulty in the application 
of the test comes in the ability to recognize the various tints of green which 
may be present. With a little practice, however, a positive reaction may be 
readily detected, especially if a control test be made with normal urine. 

This test is shown as early as the second day of typhoid fever and persists 
throughout its course. The mint-green hue is first observed, the emerald- 

1 See Umber, Med. Klin., 1912, VIII, 322; Woody and Kolmer, Arch. Pediat., 1013, 
XXIX, 18; Marcantoni, Gazz. d. osp., 1914, XXXV, 193; Jahrb. f. Kinderh., 191 5, LXXXI, 
168. 

2 See Heflebower, Am. Jour. Med. Sc, 1912, CXLIII, 221. 

3 RiformaJMedica, 1905, XXI, 507; abstract Jour. Am. Med. Assn., 1905, XLV, 363. 



33& 



DIAGNOSTIC METHODS. 



green tint appearing as the disease progresses. If the course is favorable 
the color tone becomes more and more bluish, while if unfavorable the emerald 
tint persists. This test is also given in measles, smallpox, chronic and 
suppurative tuberculosis, but is negative in varioloid, varicella, scarlet fever 
miliary tuberculosis, appendicitis, and malaria. 

This test is as simple and just as reliable as is the diazo-reaction, being 
especially valuable in differentiating a typhoid from a miliary tuberculosis. 
Rolph and Nelson 1 have pointed out that urines containing bilirubin react to 
this test, so that this fallacy must be borne in mind. 
(h). Dimethylaminobenzaldehyd Reaction. 

This test, advanced by Ehrlich, is as follows: Prepare a 2 per cent, solution 

of p-dimethylaminobenzaldehyd in equal 
parts of concentrated HC1 and water. 
Add a few drops of this solution to 5 c.c. 
of fresh cold urine and allow to stand for 
a few minutes. A positive reaction is 
indicated by the appearance of a cherry- 
red color, which may be extracted with 
chloroform or epichlorhydrin. Heating 
facilitates the reaction, but here normal 
urine may give a slight reddish coloration. 
In the cold normal urine gives a greenish- 
yellow color. Apparently this coloration 
is due to the presence of metabolic 
products derived from blood pigments 
(urobilinogen compounds, see p. 256). 2 
It is to be expected, therefore, that this 
reaction would be distinct in diseases of 
the liver and bile passages, although it is 

not constant even here. It is, also, ob- 
Fig. 89. — Purdy electric centrifuge. . . . . . 

served in tuberculosis, pneumonia, ty- 
phoid fever, malaria and scarlet fever. 3 It has little clinical value. 




IV. Microscopic Examination of the Urine. 

The microscopic examination of the urine is important in every case. 
So varied are the elements which appear in the microscopic field that con- 
siderable experience is necessary before absolute interpretation can be made. 
Not infrequently the character of the sediment will change a diagnosis, as, 
for instance, when pus- or blood-cells are present in sufficiently large amounts 

1 Medical Record, 1911, LXXX, 373. See, also, Neuman and Behrend, Arch. Int. Med., 

1913, XI, 456; da Pozzo, Gazz. d. osp., 1914, XXXV, 865. 

2 Fischer and Meyer-Betz (Ztschr. f. physiol. Chem., 1911, LXXV, 232) show that the re- 
action depends on the presence of a dipyriylphenylmethane pigment of the class of non-stable 
pyrrol derivatives, to which group the blood and biliary pigments, as well as urobilinogen, belong. 

3 See Hesse, Med. Klin., 1913, IX, 294; also, Steensma, Xederl. Tijdschr. v. Geneesk., 

1914, LVIH, 467; Rabinowitsch, Berl. klin. Wchnschr., 1914, LI, 1456; Berkowitz. Med. 
Record, 1914, LXXXVI, 1087; Eisner, Deutsch. Ztschr. f. Chir., 1915, CXXXII, 589; Robert- 
son, Calif. State Jour. Med., 1915, XIII, .65; Litzenberg (Chicago Gynec. Soc, Apr. 16, 1915) 
reports an increase of urobilinogen in the toxemias of pregnancy. 



THE URINE. 



337 



to account for an albuminuria previously determined by chemical methods. 
It is, therefore, essential that the microscopic examination of urine form a 
part of the ordinary routine. 

In obtaining the sediment of the urine for microscopic examination, 
two methods are possible. In the first place, the urine may be allowed to 
stand in a conical glass for periods ranging from 12 to 24 hours. The sedi- 
ment originally present in the urine as well as that formed by chemical changes 
taking place during the standing will collect in the lowest portion of the glass 
and may be removed by a glass-tube drawn out to a somewhat small point. 
It is to be remembered that a sediment at the end of 12 to 24 hours may be 
entirely different from that originally present in the freshly voided specimen. 
The changes in the reaction of the urine will, necessarily, lead to the dis- 
solving of certain types of crystals and to the formation of other varieties. 
Moreover, casts, if originally present, may dissolve or disappear as a result 
of the reaction of the urine becoming alkaline. For these reasons it is ab- 
solutely essential, in the use of this gravity method, that preservatives be 
added to the urine. Among the preservatives which may be used to prevent 
bacterial action during the sedimentation, we find a small piece of camphor 
or a rather large crystal of thymol serving the purpose. Some workers add 
one-fifth the volume of a 4 per cent, solution of borax which is equally useful, 
<mE== m lm but the addition of chloroform or formalin, does 
not serve as well in these cases, as the former 
does not completely preserve the casts and the 
latter introduces a crystalline compound of 
formalin and urea which may be confusing, as 
it is not unlike impure leucin. This method is 
not to be recommended for routine work, as it 
requires too long a period and as changes may 
occur which make it absolutely impossible to 
differentiate between a primary and a secondary 
sediment. 

Secondly, the sediment may be thrown 
down by the use of the centrifuge. This ap- 
paratus is seen in the accompanying cut. By 
the use of this method no preservative is needed, 
a deposit is obtained within three minutes in a 
much more concentrated 'form, and changes in 
the sediment do not take place. The writer 
would recommend, therefore, the use preferably 
of the electric centrifuge or, at least, of the type 
run by hand. 

Whatever method may be adopted for 
obtaining the urinary sediment, the next steps in the process are the same. 
A pipet, consisting of a glass tube drawn out to a point about one-half the 
diameter of the tube, is introduced to the bottom of the vessel containing the 
sediment, a finger being placed over the upper end to prevent fluid passing into 



Fig. 90. — 
Sediment tube 
for Purdy cen- 
trifuge. 




Fig. 91= — Per- 
centage c e n t r i - 
fuse tube. 



338 DIAGNOSTIC METHODS. 

the tube as it is introduced. When the tip of the pipet comes in contact with 
the deposit, the pressure of the finger on the upper end of the pipet is removed 
and the deposit allowed to flow up into the pipet. The finger is then placed 
tightly over the tube as it is withdrawn from the fluid. By placing the tip of 
the pipet in contact with a perfectly clean slide which is absolutely free from 
scratches and by gradually rotating the pipet, a small portion of the sedi- 
ment is obtained. A cover-glass is then placed upon this drop avoiding any 
undue pressure, which might distort the organized elements of the sediment. 
Some workers dispense with the cover-glass and use somewhat larger amounts 
of sediment, but the writer does not find this method as acceptable owing 
to the fact that the lens of the objective may dip into the fluid and thus give 
indefinite microscopic pictures. Moreover, the focus cannot be as accurately 
adjusted without the use of the cover-glass. 

In the examination of the microscopic specimen, prepared as above, 
the point of special importance to be observed is the proper adjustment of the 
light. The writer is accustomed to use the low-power objective in the pre- 
liminary examination. In this case it is essential that the light be shut off 
to a large extent, as the recognition of casts cannot be made in a brilliantly 
illuminated field. This examination with the low power has the advantages 
that larger visual fields are subject to inspection, casts are easily recognized 
and crystalline deposits, as well as morphological elements, are usually differen- 
tiated. After the preliminary examination with the low power, the final 
examination is made with the high-power dry lens. In this way elements 
which appear suspicious under the low power are more clearly brought out 
and differentiations made possible between various types of cellular elements. 
With the high-power lens it is, of course, essential that the field be somewhat 
more illuminated than when the low-power is used. While a mechanical 
stage is, at times, advantageous in the microscopic examination of the urine, 
the writer has found that the fingers serve practically every purpose in the 
manipulation of the slide under examination. 

Where the urine is to be examined for the presence of bacteria, stained 
specimens must be made and examined with the oil-immersion lens. 

Urinary sediments are classified into two divisions: (a) chemical or 
nonorganized and (b) anatomical or organized sediments. The nonorganized 
sediments exist in solution in normal urine and appear as deposits under 
conditions of excessive formation, excessive excretion, or of alterations in the 
urine affecting its solvent properties. The chief . chemical sediments are 
uric acid and its salts, calcium oxalate, phosphates, sulphates, cystin, leucin, 
tyrosin, xanthin, fat, and fatty acids. The . organized sediments are usually 
foreign substances and are not met with in normal urine. They consist of 
epithelial cells, pus corpuscles, blood-cells, renal casts, spermatozoa, infusoria, 
bacteria, and tissue fragments. 
(^4). Unorganized Sediments. 
(a). Those Appearing in Acid Urine. 

(1). Uric Acid (C s H 4 N 4 3 ). : 

This occurs as a sediment in the urine under 3 conditions: (1) great 



THE URINE. 



339 



concentration, (2) high acidity, and (3) low temperature. The deposit differs 
from others in possessing a deep yellow or orange-red color, although some 
of the smaller crystals are occasionally colorless. The primary form of the 
uric acid crystal is that of the rhombic prism. Modifications of this, in the 
form of square plates, cubes, ovoids, dumb-bells, or whetstone crystals are 
sometimes noticed. A rare type, especially of the colorless crystals, is a per- 
fect hexagon which resembles cystin so closely that chemical means of differen- 
tiation must be used. The crystals may be single or grouped in rosettes or 
fan-shaped masses. Occasionally typical needle-shaped crystals may be seen 
which are arranged in sheaves. 

The microscopic peculiarities of uric acid are usually such as to permit 
of its easy recognition. In some cases, however, it is wise to confirm the micro- 




Fig. 92. — Various forms of uric acid. 1, Rhombic plates; 2, whetstone forms; 3, quadrate 
forms; 4, 5, prolonged into points; 6, 8, rosettes; 7, pointed bundles; g, barrel forms pre- 
cipitated by adding hydrochloric acid to urine. (Hawk.) 



scopic finding by the murexid test as follows: Place a small quantity of the 
sediment in an evaporating dish and add a few drops of concentrated nitric 
acid. Evaporate on the water-bath to dryness, when a yellowish or reddish 
residue will remain. Allow the residue to cool and add a few drops of 
ammonium hydrate solution. In the presence of uric acid a distinct reddish 
purple color will appear. If water be added to this purple solution and the 
mixture evaporated to dryness the color disappears. This latter point is 
of importance as xanthin, which may resemble unusual types of uric acid in 
microscopic appearance, also gives the murexid test, but the color does not 
disappear on heating with water. 

(2). Sodium Acid Urate (C 5 H 3 NaN 4 3 ) . 

This salt of uric acid forms the bulk of the " brick-dust deposit" or "sedi- 
mentum lateritium" found when urine has cooled. In such cases the urine 



34o 



DIAGNOSTIC METHODS. 



first shows a milky appearance and the sediment soon settles on the sides 
and bottom of the container. This deposit is usually in the form of irregular 




Fig. 93. — Acid sodium urate. {Hawk.) 

amorphous granules of a brownish or pink color. Occasionally the sediment 
may be distinctly crystalline, occurring as prismatic needle-like crystals which 
are grouped in star-shaped, fan-shaped or dumb-bell-like clusters. 




Fig. 94. — Xanthin. {Hawk.) 

(3). Potassium Acid Urate (C S H 3 KN 4 3 ). 

This substance occurs only as a granular amorphous deposit. Owing 
to its greater solubility, it does not form as large an amount of the brick-dust 
deposit as does the sodium salt. 

These two latter sediments are occasionally associated with amorphous 
deposits of the calcium and magnesium acid urates. These are, however, 
rare and need not be separately considered. In detecting the presence of the 
urates in a deposit, a small portion of the turbid urine is poured into a test-tube 
and gently heated. If urates are present the sediment will completely dissolve. 
These salts also give the murexid test. 



THE URINE. 341 

(4). Xanthin (C 5 H 4 N 4 2 ). 

This substance which is chemically closely related to uric acid is rarely 
found as a sediment in the urine. Its chief clinical importance is found in 
its appearance as a urinary calculus. It crystallizes in whetstone-shaped 
colorless crystals which resemble those of uric acid, from which it is differen- 
tiated by its solubility on heating and in hydrochloric acid as well as ammonia. 
It may be chemically recognized by WeideVs reaction. Place a portion of 
the suspected crystalline deposit in an evaporating dish, and dissolve by warm- 
ing with a few drops of bromine water. Evaporate to dryness and place the 
dish containing the residue under a large beaker, allow the fumes of ammonia 
to fill the inverted beaker when a red or purplish-violet color will be produced 
in the presence of xanthin. 

(5). Calcium Oxalate (CaC 2 4 ). 

This substance appears most frequently in acid urine, but may be found 




« 



Fig. 95. — Calcium oxalate. {Hawk after Ogden.) 

after the urine has undergone alkaline fermentation. If it occurs in acid urine 
it is associated with uric acid; if in alkaline urine, with the triple phosphates. 
The deposit is a colorless crystalline one having two distinct forms: (1) octahe- 
dral crystals (four-sided pyramids lying base to base) ; viewed from the side, 
these appear as squares crossed by two sharp lines, giving the so-called "en- 
velope" crystal. (2) Dumb-bell crystals in the form of ovoid or circular 
disks with round margins depressed at the centers. These latter often present 
radial striations. Emerson has called attention to a rare type of calcium 
oxalate crystal which appears in the form of flat plates with parallel sides 
and rounded ends, looking like superimposed sheets of mica. It is character- 
istic of the crystallization of this substance, as of most crystalline urinary 
deposits, that the crystals are practically always of the same type, variations 
rarely appearing in the same specimen of urine. 

These crystals are insoluble in acetic acid, but soluble in hydrochloric 
acid. This is a point of some importance, as it is occasionally difficult to 
distinguish microscopically between calcium oxalate and some crystals of 
triple phosphate. This latter crystal is soluble in acetic acid. These crystals 
may be chemically identified by dissolving them in hydrochloric acid, alkaliniz- 
ing with ammonium hydrate, and precipitating with ammonium oxalate. 



342 DIAGNOSTIC METHODS. 

(6). Cystin (C 3 H 6 NS0 2 ) 2 . 

The appearance of cystin in the urine is known as cystinuria. This is 
a condition of perverted protein metabolism which is not well understood. 
It may make its appearance at any period of life and in either sex, perhaps 
somewhat more frequently in the male. It shows a remarkably frequent 
hereditary character, being observed in some cases through several genera- 
tions and in several children of the same parents. Many of these cases do not 
show any clinical characteristics, being present during their entire life without 
any apparent symptoms. In other cases, owing to the formation of calculi, 
frequent manifestations are noted and surgical intervention interposed. 
This condition is not very frequent, being reported only 180 times in the 
literature. 1 

" Prior to the time when cystin was found to be a product of the disintegra- 
tion of protein substances it had been conjectured, on the ground of its content 
in sulphur and nitrogen, that it might be a product of the intermediate pro- 
tein metabolism. The explanation which von Udranzki and Baumann gave 



o 



I 




o 




o 

Fig. 96. — Cystin. (Hawk after Ogden.) 

of its excretion corresponds in principle to that which is familiar to-day for 
the appearance of conjugation products of glycuronic acid and of glycocoll 
in urine. Like the latter, it should be normally further oxidized, and only 
in the presence of definite bodies should it be intercepted. These authors 
considered the binding substances to be the previously mentioned diamins, 
especially putrescin and cadaverin, which they had found in the urine and 
in the feces of a cystinuric patient. The formation of the diamins was sup- 
posed to be brought about by specific bacteria in the digestive tract by an ex- 
traordinary chronic intestinal mycosis. The resorbed part of the cadaverin 
and putrescin was supposed to protect the cystin from combustion, just as 
benzoic acid does glycocoll, by entering into a loose combination which de- 
composes again after passing through the kidneys. Serious difficulties, how- 
ever, have opposed themselves to this interpretation of cystinuria. First, 
numerous cases have been described without simultaneous diaminuria, and, 
conversely, diaminuria occurs in malaria, and in the conditions brought about 
by the cholera vibrios and the Finckler-Prior bacillus, without cystinuria 
having ever set in, any more than it does when diamins themselves are 
administered." 

1 See Fromherz, Berl. klin. Wchnschr., 1913, L, 1618; Neumann, Deutsch. med. Wchn- 
schr., 1914, XL, 2065. 



THE URINE. 



343 



"Further investigations have shown that cystinuria is really a disturbance 
of the amino-acid metabolism. Of the end-products of protein hydrolysis 
which arise in the system, the cystinuric cannot avail himself in the normal 
way of the cystin, and in part excretes it; the remaining products of protein 
hydrolysis undergo their ordinary fate. If free monomolecular a-amino 
acids appear in places which are at present not well known, or if they occur 
there even in unusual amounts, then, unlike the normal individual, the cystin- 
uric is unable to burn them and they leave the organism unchanged just 
as cystin itself does. The basic diamino acids behave in practically the same 
way, except that the C0 2 group is split off from them and we arrive at diam- 
inuria" (Neuberg). 

Crystals of cystin are rare in the urinary sediment. In some of the cases 
reported the cystin did not separate from the urine until this was acidified with 
acetic acid and allowed to stand for 12 hours. It crystallizes in two forms: 




Fig. 97. — Pure leucin. {Hawk.) 

(1) six-sided tablets having an opalescent luster and sometimes traced with 
fine lines of secondary crystallization; (2) four-sided square prisms lying 
separately or in stellat$ forms. These crystals are soluble in hydrochloric 
acid, alkaline hydrates, and insoluble in acetic acid. These tests differentiate 
it from uric acid. If the urinary sediment suspected of containing cystin be 
treated with strong sodium hydrate solution and a few drops of benzoyl chlorid, 
and the mixture shaken, a voluminous precipitate' of benzoyl cystin is obtained. 

(7). Leucin (C 6 H 13 N0 2 ). 

Chemically leucin is a-aminoisobutylacetic acid. It occurs in the 
urine in conditions associated with more or less marked derangement of hepatic 
function (see amino acids). As found in the urine, leucin appears in the form 
of yellowish, highly refractile spherules, with alternating light and dark 
concentric layers and with radial striations. In the pure state it crystallizes 
either in thin, white, hexagonal plates or as scales or rosettes of irregular 
shapes. 



344 



DIAGNOSTIC METHODS. 



Leucin is soluble in water, acids, and alkalies, and insoluble, to a more 
or less extent, in alcohol. Not always do we find crystals of leucin in the sedi- 
ment when the urine contains this substance. If it be suspected, the urine 
should be evaporated to a small bulk and alcohol added to the residue, which 
may then be examined for the characteristic crystals. This leucin may be 
identified by Scherer's test as follows : Some of the solid residue obtained by 
concentrating the urine to a small bulk is evaporated with concentrated nitric 
acid on a platinum crucible cover. With pure leucin the residue remains 




Fig. 98. — Impure leucin. {Hawk after Ogden.) 

colorless, but as usually applied to the urine a yellowish residue obtains. This 
is heated with a few drops of sodium hydrate solution, when a yellowish or 
brown color will be observed. If further heating be applied the leucin will 
collect into an oily drop which rolls around on the heated surface. As leucin 
does not stain with Sudan-Ill it should not be confused with fat. 

(8). Tyrosin (C 9 H n N0 3 ). 

Chemically, tyrosin is p-oxyphenyl-a-amino-propionic acid. As found 




Fig. 99. — Tyrosin. (Hawk.) 

in the urine tyrosin crystallizes in the form of fine colorless needles, which 
may appear black and are arranged in sheaf-like collections or rosettes. Like 
leucin, it may not crystallize out unless the urine be concentrated. Tyrosin 



THE URINE. 345 

is soluble in water, acids, and alkalies, while it is slightly soluble in alcohol 
and insoluble in ether. As other crystals, which may appear in the urine 
closely resemble the tyrosin needles, it is advisable to confirm the microscopic 
findings by chemical tests. 1 This may be done by evaporating the urine to a 
small bulk, removing the fluid and dissolving the residue in water. Morner's 
test may then be applied as follows: To this aqueous solution is added i c.c. 
of a reagent consisting of i c.c. of formalin, 55 c.c. of concentrated sulphuric 
acid, and 45 c.c. of water. If the mixture be heated to boiling a beautiful 
green color will be observed in the presence of tyrosin. 

(9). Calcium Sulphate (CaS0 4 2H 2 0). 

This is a very rare sediment, appearing only when the urine is extremely 
acid. The crystals appear in the form of long, thin, rhombic plates or needles 
which may be single, but are more frequently observed in clusters. If the 
sediment be boiled with hydrochloric acid and barium chlorid added, a 
precipitate of barium sulphate will point to the presence of calcium sulphate 
in the sediment. 








Fig. 100. — Calcium sulphate. (Hawk Fig. ioi. — Bilirubin (Haematoidin). 

after Hensel and Weil.) (Hawk after Ogden.) 

(10). Bilirubin (C 16 H 18 N 2 3 ). 

Bilirubin or its isomer hematoidin may appear in the urine in conditions 
previously discussed. The type of crystal is either a brilliant yellow or ruby- 
red rhomb or a yellow needle. Rarely the deposit may be in the form of a 
yellow granular sediment. Not infrequently small curved needle-like spines 
are observed projecting from the angles of the rhombic' crystals. These 
crystals may be identified by extracting the acid urine with chloroform and 
applying the tests previously discussed under Biliary Pigments. 

(11). Hippuric Acid (C 9 H 9 N0 3 ). 

This substance has been observed as a sediment, although rarely. It ap- 
pears in the form of semitransparent, colorless, four-sided prisms, or in long 
pointed rods or needles, occasionally in forms closely resembling those of the 
triple phosphates to be described later. These crystals are soluble in warm 
water, alcohol and ether and may be distinguished from uric acid by the fact 
that they do not give the murexid test. 

1 See Rosenbloom and Gardner, New York Med. Jour., 1914, C, 574. 



346 DIAGNOSTIC METHODS. 

(12). Neutral Calcium Phosphate (CaHP0 4 2H 2 0) . 

This substance is found only in faintly acid or neutral urine. It is quite 
rare as a sediment, crystallizing in colorless needles or slender pyramids which 
group themselves together with their points in a common center to form rosettes 
or cross-shaped figures. These crystals are soluble in acetic acid and may 
be converted into calcium carbonate when treated with a strong solution of 
ammonium carbonate. 

(13). Fat. 

Under normal conditions the urine contains no free fat, but amounts 
varying from traces to rather large excretions may be found under pathologic 
conditions. 1 The excretion of fat in the urine is known as lipuria. This is 
characterized by the presence of small or large strongly refractile globules 
which may be stained black with osmic acid or red with Sudan-Ill. These 
globules are soluble in ether and may, therefore, be extracted from the urine 
by shaking out with this solvent. It not infrequently happens that the urine 
is contaminated with fat which may have been used in obtaining catheterized 
specimens or with fat coming from the bottle containing the urine. This 
may lead to a diagnosis of lipuria unless care be taken to exclude such a source. 
True lipuria has been observed in various conditions. Thus we may find 
after a large intake of fat in the diet or as a therapeutic agent the so-called 
"alimentary lipuria" Pathologically, it has been observed in various cachectic 
conditions, in crushing injuries, especially of the bones, in eclampsia, in chronic 
heart disease, fatty tumors, diabetes mellitus, tuberculosis, various affections 
of the pancreas and liver, nephritis, and after the use of various general 
protoplasmic poisons. In these cases the blood may also contain an excess of 
fat, although this has not been observed in all cases. In fatty degeneration 
along the genitourinary tract, fat droplets may be seen in the epithelial cells 
and in the casts. Free fat is rarely found in such conditions, but occasion- 
ally it may collect in droplets which float on the surface of the urine and then 
constitutes true lipuria. Occasionally flat superimposed plates with notched 
corners (cholesterin) may be seen and may be so numerous as to justify the 
term "cholesterinuria." 

In conditions associated with infection by the filaria, large amounts 

of fat may be present, giving rise to the appearance of an emulsion. To this 

condition has been given the name chyluria. In this form the fat may be 

present occasionally in large masses resembling tallow, but more frequently 

is seen in finer clumps of globules. The appearance of the urine is much 

like that of skimmed milk, but may have a reddish tinge due to the presence 

of blood. On allowing the urine to stand, a cream-like mass of fat will rise 

to the surface. It is not unusual in such cases to find the ova or the parasite 

in the masses of coagulated material. The excretion of the fatty material 

at times runs a somewhat cyclic course, being present during the day and 

absent at night or vice versa. Occasionally the excretion varies with the 

position of the patient, being somewhat more frequent when he is erect, and 

1 See Sakaguchi, Biochem. Ztschr., 1913, XLVIII, 1; Lawrynowicz, Ztschr. f. klin. Med., 
1914, LXXX, 389. 



PLATE X. 




Ammonium Urate, showing Spherules and Thorn-apple-shaped Crystals. 
(From Ogden, after Peyer.) 



THE URINE. 347 

may be markedly increased after severe exercise. This condition should be 
taken simply as a symptom of filariasis. 

A nonparasitic type of chyluria has been observed, but its etiology is 
somewhat uncertain. It probably is closely related to the conditions above 
mentioned as causing true lipuria. It does have some relation to an increased 
fat diet and apparently is associated with exudation from the lymphatic vessels, 
as the cellular elements are largely lymphocytes. 1 




Fig. 102. — Cholesterin. (Hawk.) 

(b). Those Occurring in Alkaline Urine. 

(i). Ammonium Urate (C S H 3 (NH 4 )N 4 3 ). 

This sediment occurs most frequently in combination with amorphous 
calcium phosphate and triple phosphate crystals. It is the only urate deposit 
found in alkaline urine, but may occur, in neutral urine. It appears as a 
crystalline deposit of dark brown spherical masses studded with fine spiculae, 
from which fact the name of " thorn-apple " crystals has been given to them. 
Occasionally these spheres may show concentric or radial striations. Not 
infrequently one observes crystals having irregular shapes, such as those of 
a dumb-bell or a pear. 

Chemically, these crystals may be identified by dissolving in hydrochloric 
acid, when uric acid, which may be identified by the murexid test, will separate. 
If sodium hydrate be added to the dry sediment and heat applied, vapors 
of ammonia are given off. 

(2). Calcium Triphosphate (Ca 3 (P0 4 ) 2 ). 

This compound is frequently found in alkaline urine, as a white amorphous 
flocculent deposit arranged in irregular patches. This is the usual deposit which 
appears in the urine when it becomes alkaline after meals. In the so-called 
" phosphaturia" the urine is always turbid when voided so that the assumption 
was made that an excess of phosphoric acid was being excreted. Such is 

1 See Stern, Arch. Diagnosis, 1913, VI, 114; Young, Jour. Trop. Med. andHyg., 1914* 
XVII, 241. 



348 DIAGNOSTIC METHODS. 

found not to be the case, as a deposition of the normal phosphates of the urine 
must occur when the reaction becomes alkaline. It is to be said in this place 
that no conclusion whatever can be drawn from the separation of a substance 
in the sediment as regards an increase in its excretion. So many factors 
influence the separation or nonseparation of a sediment that a finding should 
not be regarded as evidence of increased formation and excretion unless quan- 
titative chemical examination points in this direction. 

Calcium phosphate is soluble in acetic acid without evolution of gas, 
which test may be used to show the presence of this substance in the deposits. 
It may be absolutely identified as a calcium compound by dissolving in acetic 
acid and precipitating with ammonium oxalate; the phosphoric acid radical 
may be proven by dissolving the sediment in nitric acid and precipitating with 
ammonium molybdate. 

(3). Magnesium Phosphate (Mg 3 (P0 4 ) 2 ). 

Theoretically this compound appears along with calcium phosphate 
as an amorphous deposit in alkaline urine. Its amount is, however, usually 
less than the latter compound. 

It is observed in rare cases as large, long, rhombic plates with beveled 
edges which closely resemble the crystals of triple phosphates. These crystals 
are found in cases in which not sufficient ammonia is present to form the true 
triple phosphate, and may be considered as transition crystals. 

(4). Magnesium-ammonium Phosphate (Mg(NH 4 )P0 4 ). 

The appearance of this substance in the urine is essentially characteristic 





Fig. 103. — Magnesium-ammonium phosphates. {Hawk after Ogden.) 

of ammoniacal urine. It may very rarely be seen in amphoteric urine when 
ammonium salts are present in large amounts. The crystals belong to the 
rhombic system, appearing most frequently as triangular prisms or "coffin- 
lid" crystals. These may be shortened in the form of squares or one or more 
corners may be rounded or beveled. By refracted light a greenish tone is 
observed when these crystals are present. A second type of the "triple phos- 
phate" is that of a star-shaped feathery crystal with points not unlike fern 
leaves. These crystals are easily soluble in acetic acid and may be identified 
by treating with sodium hydrate and warming when ammonia is evolved. 



THE URINE. 349 

(5). Calcium Carbonate (CaC0 3 ). 

This substance frequently occurs in alkaline urine in association with 
the amorphous phosphates. It may appear as groups of amorphous material 
or may form large spheroidal masses with concentric radiations. Occasionally 
it may be observed in dumb-bell like masses which resemble somewhat the 
same type of calcium oxalate crystal, from which it may be differentiated 
by the fact that it is soluble in acetic acid with production of C0 2 , while calcium 
oxalate remains undissolved. 




Fig. 104. — Calcium carbonate. {Hawk.) 

(B). Organized Sediments. 
(1). Mucoid material. 

Mucus is a constituent of practically every specimen of urine, in the 
form of the "nubecula." This appears in the form of small threads which 
branch and interlace in such a way that the entire microscopic field may be 
practically taken up by this material. In the meshes of the nubecular threads 
are observed the so-called "mucous corpuscles," which are practically identical 
with the ordinary leucocyte. Little significance is attached to this form of 
mucous threads unless a great increase is observed, when it indicates, as does 
mucin, a vesicle catarrh. 

(2). Epithelial Cells. 
Normally, the only epithelial cells found in the urine are the irregular 
flat cells from the bladder and urethra or the large flat epithelia seen in the 
urine of women and arising from the vagina. The presence of large num- 
bers of other types of epithelial cells is always pathological and denotes an 
inflammatory or destructive lesion somewhere along the genitourinary tract. 1 
It is a matter of more or less difficulty absolutely to identify, in all cases, the 
source of the epithelium found in the urine. According to Heitzmann, the 
positive recognition is based largely upon the size of the cell, as the shape 
may vary from pathologic conditions as well as from the portion from which 

1 See Schkarin, Russk. Vrach. 1915, XIV, 505. 



35° 



DIAGNOSTIC METHODS. 



they are derived. As stratified epithelium is found in the pelvis of the kidney, 
the ureters, bladder, and urethra, it is to be expected that large flat cells, 
cuboidal cells, or columnar cells will appear depending upon the layer from 
which the cell is derived. As the simple epithelium exists in the uriniferous 
tubules, the prostate gland, seminal vesicles, and ejaculatory ducts, the recog- 
nition of such cells will limit their origin, the size being important in determin- 
ing the exact point from which they are derived. It is to be remembered, 
therefore, that the shape of the cell is of far less importance than is its size. 

The epithelial cells derived from the bladder are usually the large flat 
irregular cells commonly seen in all normal urine. They have a clear pro- 
toplasm and usually a small distinct central nucleus and are extremely granular. 
These flat epithelial cells may be single, in groups, or if the irritation is marked 




Fig. 105. — Epithelium from different areas of urinary tract, d, leucocyte (for com- 
parison); b, renal cells; c, superficial pelvic cells; d, deep pelvic cells; e, cells from calices; 
/, cells from ureter; g, squamous epithelium from bladder; h, neck of bladder cells; i, epi- 
thelium from prostatic urethra; k, urethral cells; /, scaly epithelium; m, m 1 , cells from seminal 
passages; n, compound granule cells; 0, fatty renal cell. (Hawk after Ogden.) 



may occur in large sheet-like masses. The large cuboidal cells of the bladder 
epithelium may be seen in acute cystitis in which they are associated with large 
numbers of the flat cells previously mentioned. If the conditions become 
chronic the flat cells may entirely disappear and be replaced by cuboidal and 
by a few columnar epithelial cells. These latter cells are especially observed 
in the severe inflammatory processes in the bladder. 

The large, flat, squamous epithelial cells derived from the vagina are 
more frequently arranged in stratified groups so that their recognition is usually 
simple. As these types of cells denote simple desquamation, being pathologic 
only when present in enormous numbers, an absolute differentiation is of 
little consequence clinically and, if it be so, the clinical symptoms of the case 
will usually clear up the decision. 

The urethral epithelium very closely resembles that above described. 



THE URINE. 351 

The cells are large and irregular, being partly flat, partly cuboidal and partly 
columnar. The cylindrical types of urethral epithelium may occur in the 
form of longer, irregular, smaller types, than those of the bladder or vagina. 
This type constitutes the so-called "tailed" cells, which may be derived from 
the pelvis of the kidney and were at one time held to be indicative of a pyelitis. 
Sahli regards a preponderance of such tailed cells over the flatter and more 
regular types as distinct evidence of trouble in the renal pelves. As these 
cells may be derived from other portions of the urinary tracts it is unwise 
to make an absolute diagnosis on such a finding. The small polygonal cells 
as well as tailed cells may be derived also from the ureter so that our diagnosis 
would necessarily rest upon findings other than such epithelium. 

In the writer's opinion, it is a practical impossibility to make a positive 
diagnosis of a lesion in any specific portion of the genitourinary tract based 
entirely upon the appearance of the urinary epithelium. The points to be 
remembered are that we may have any type of epithelium and may have many 
variations in shape as well as in size. Such variations may be present in any 
portion of the urinary system, although distinctly renal epithelial cells are 
more frequently in the form of round or cubical cells somewhat larger than 
the leucocyte and containing a large vesicular nucleus. These latter renal 
cells are the only ones which seem to the writer distinctly diagnostic. They 
are differentiated from the similar cells arising from the ureters and prostate 
gland by the fact that the latter cells are about twice the size of the pus-cell, 
being consequently larger than the true renal cell. 

Degenerative changes are frequently observed in these epithelial cells, 
even when examined immediately after voiding. The usual type of this de- 
generation is the presence of fat granules or globules, especially in the small 
renal cells. If the sediment be treated with Sudan-Ill these granules will 
appear distinctly red. 

(3). Pus-cells. 
A few leucocytes may be observed in practically every specimen of urine, 
especially in those from women, in which case they may be in large numbers 
and derived from the vagina. A marked increase, as recognized by numerous, 
indistinct, small, circular or irregular, granular cells, should be regarded as 
pathologic. 1 To this condition the name pyuria has been given. The simple 
finding of a pyuria does not necessarily indicate the point from which these 
cells were derived. Severe inflammatory processes anywhere along the 
genitourinary tract or the rupture of an abscess into the urinary tract will 
be associated with a pyuria, so that other features must be relied upon in 
deciding as to the source. As a rule, it may be said that the amount of pus 
is small in direct affections of the renal cortex, while disease of the urinary 
passages is associated with a larger number. If an abscess has ruptured 
into the pelvis of the kidney the number of cells may be enormous. If the 
pyuria be of renal origin, it will be associated with the presence of the small, 

1 See Posner, Arch. Diagnosis, 1912, V, 269; also, Pedersen, New York Med. Jour., 19 13 , 
XCVIII, 1 14 1. 



352 



DIAGNOSTIC METHODS. 



round, renal epithelial cells as well as with tubular casts. Frequently leuco- 
cytes in small numbers are found adherent to the casts or they may even be 
so closely grouped as to give the name pus cast to such formations. If large 
numbers of pus-cells appear in the course of a chronic nephritis, they indicate 
either an acute exacerbation of the condition or a complicating process in some 
other portion of the urinary tract. The sudden appearance of very large 
numbers of pus-cells is especially indicative of a ruptured abscess. In inflam- 
matory processes in the pelvis of the kidney the amount of pus may vary 
within wide limits. In some cases the urine may be perfectly clear when 
voided, showing the presence of only a few pus-cells, while in others enormous 
numbers may appear. This paradoxical condition may be accounted for 
by the possibility of obstruction of the ureter on the affected side and the later 




Fig. 106. — Pus corpuscles, i, Normal; 2, showing ameboid movements; 3, nuclei 
rendered distinct by acetic acid; 4, as observed in chronic pyelitis; 5, swollen by ammonium 
carbonate. {Hawk after Ultzmann.) 

forcing out of the large numbers of pus-cells. In pyelitis the urine is usually 
acid, which may serve as a distinguishing point from cystitis in which the 
urine is almost always alkaline. 

In tuberculosis of the renal parenchyma pus-cells appear very early and 
vary in number from a few to many thousands. This pyuria is usually con- 
stant, and is frequently associated with hematuria. The pus-cells in tubercu- 
losis are usually of the mononuclear type instead of the ordinary polymorphonu- 
clear form. This is not easily determined, as the degenerative processes make 
it somewhat difficult to distinguish the nuclear form. In such conditions 
the sediment should be frequently examined for the presence of tubercle 
bacilli and a portion inoculated into a guinea-pig. This is the only certain 
method of making a diagnosis of tuberculosis of the kidney. It is, perhaps, 
needless to add that for absolute differentiation a specimen obtained by 
ureteral catheterization must be examined. 

In cystitis the number of pus-cells appearing in the urine will vary ac- 



THE URINE. 353 

cording to the severity of the condition, the more severe the more pus-cells. 
In this condition the urine is alkaline and may be, when voided, glairy and 
ropy. In chronic cases of cystitis the pus-cells, although present in the bladder 
in large numbers, may be so degenerated by the alkalinity of the contents 
that practically no cells are recognizable. Here we find the appearance 
of a large amount of mucus, the urine being in some cases distinctly jelly-like. 

In inflammatory processes of the urethra pus may be present in varying 
amounts. In the acute conditions the number of cells is much more numerous 
than in the chronic types. The recognition of the causative factor, in most 
cases the gonococcus, will be treated of in a later section. As the acute condi- 
tion becomes subacute or chronic, the urine contains large numbers of the 
so-called gonorrheal threads which enclose numerous pus-cells. 1 These will 
be treated in detail later. It is sometimes a matter of clinical importance to 
distinguish between an anterior and a posterior urethritis. This is best done 
by the so-called "two-glass" test. If the first portions of the urine be collected 
in a receiving vessel and the later portions in a second vessel, the urine in 
the first vessel will be cloudy while that in the second vessel is clear in the case 
of anterior urethritis; while in posterior urethritis associated with the anterior 
type the first portion will be cloudy and the second usually so, although at 
times it may be clear. The reaction of the urine in both vessels will be acid 
unless a complicating cystitis has arisen, when the urine in the second vessel 
will usually be alkaline. 

The appearance of the pus-cells will vary depending upon the reaction 
of the urine. In acid urine their structure is very well preserved, the addition 
of acetic acid rendering the nucleus somewhat more distinct. Their usual 
form is that of the polymorphonuclear neutrophile, their size varying from 
7 to 12 microns. If stained the vesicular character of the nucleus of the renal 
epithelial cell will absolutely differentiate it from the irregular type of pus-cell. 
In alkaline urine the cells swell up, lose their shape and become opaque. 
The addition of acetic acid usually clears them in such a way that the nucleus 
becomes visible, but occasionally does not. If the urine remains long in con- 
tact with the alkaline material in the bladder it becomes slimy, stringy, and 
gelatinous, owing to its large content in mucus. Albumin is always present 
so that it may be difficult to decide whether or not a true albuminuria exists. 

As the pus-cells may undergo such marked change when in alkaline urine 
and be converted entirely into a gelatinous mass in which corpuscles cannot 
be detected, certain tests must be applied for positive recognition of pus in 
such cases. 

Vitali's Test. 

Acidify the urine with acetic acid and filter. Treat the material on 
the filter with a few drops of tincture of guaiac, when a deep blue color will 
appear in the presence of pus. If the material is not filterable, as happens 
when the purulent material is extremely gelatinous, place a portion of this 
slimy urine in a test-tube and allow a few drops of tincture of guaiac to flow 

1 See Broadman, New York Med. Jour., 1913, XCVIII, 28; also, Thomas, Am. Jour. 
Med. Sc, 1913, CXLVI, 696. 

23 



354 DIAGNOSTIC METHODS. 

upon the surface. If pus be present a distinct blue line of contact will be 
observed. 

Donned Test. 

A portion of the urinary sediment in a centrifuge tube is treated with 
a few drops of concentrated solution of sodium hydrate. If pus be present 
an extremely viscid gelatinous mass will be obtained. If this mixture be 
heated, it will dissolve, according to Miiller, with the formation of £-nucleinic 
acid. 

If the pus-cells be treated under the microscope with a few drops of Lugol's 
solution, they will take a mahogany-brown color owing to the presence of 
glycogen. 

Enumeration of Pus -cells. 

Such a procedure is not a part of the ordinary routine examination of 
urine. It is, however, sometimes advisable, as it permits of a decision regard- 
ing the presence or absence of a true albuminuria. If the latter exists, casts 
and renal epithelial cells will usually be present so that a diagnosis is often 
possible without a count of the cells. 

Technic. 

A portion of the 24-hour specimen of urine is thoroughly shaken to bring 
the corpuscles into suspension. This turbid fluid is then drawn up to the 
upper (11) mark of the leucocytometer, a drop placed upon the glass slide 
and the cells counted as described under Blood. If more than 30,000 per cmm. 
are present, it is advisable to dilute five times with 3 per cent, sodium chlorid 
solution. For each 100,000 leucocytes per cmm. of urine 0.1 per cent, of al- 
bumin is assumed to be present, according to Wunderlich. 

(4). Red Blood-cells. 

The presence of red blood-cells in the urine is known as hematuria. This 
condition should be sharply differentiated from hemoglobinuria as the clinical 
significance is entirely distinct. Blood may be found in the urine in a variety 
of conditions. Thus in the more malignant types of the acute infectious fevers 
hematuria is frequently observed. Likewise, in scurvy, hemophilia, purpura, 
leukemia, and Werlhof's disease, the kidney may be so markedly affected 
that hematuria obtains. 

In the hematuria of purely renal origin we find both acute and chronic 
congestions as well as inflammatory processes in the kidney associated with 
this condition. 1 In the more acute types of nephritis, hematuria is so common 
that the name "hemorrhagic nephritis" is frequently applied. Such cases 
are especially observed after poisoning with cantharides and phenol deriva- 
tives. 2 The chronic parenchymatous type of nephritis is, according to Weigert, 
always hemorrhagic in type, the number of red corpuscles being an indication 
of the intensity of the process. In malignant growths of the kidney, tuber- 

1 See Randall, Jour. Am. Med. Assn., 1913, LX, 10. 

2 See Belkowski, Rev. de med., 1913, XXXIII, 663, for a discussion of hematuria caused 
by urotropin. 



THE URINE. 355 

culosis, renal calculus, and cystic degeneration of the kidney, hematuria 
is especially common; while in infection with certain parasites, such as the 
filaria, echinococcus, and the distoma hematobium, hematuria is relatively 
frequent although few cases of these conditions are seen. 

Hematuria may also be observed as a result of lesions or disease of 
any portion of the urinary tract. Thus stone in the ureter or urethra, tumors, 
ulcers, and parasites of the bladder, urethritis, prostatitis or injury during cathe- 
terization may also be associated with the appearance of red cells in the urine. 

A further type of cases in which hematuria occurs is known as the functional 
or idiopathic hematuria. In this class of cases no definite lesion has been 
found to account for the condition. It has been called " Gull's renal epistaxis," 
"essential renal hematuria," "angioneurotic hematuria," "renal hemophilia" 
and "renal aneurysm." The lesion, whatever it may be, is usually unilateral 
and the attacks appear at variable intervals. Some of the cases recover with- 
out any treatment after one or two profuse hemorrhages, while others require 
extensive treatment of the nervous system. 1 

In the diagnosis of a hematuria it is important to observe the appearance 
of the urine both with the naked eye and with the microscope. The urine 
is turbid, and varies from a light, hazy, "smoky" appearance to a bright- 
red or deep-brown color. The red cells appear in various stages of preserva- 
tion. In some cases the normal yellow color of the cell will be quite distinct 
while in others the color will be entirely washed out. If the urine be par- 
ticularly concentrated many crenated forms will also be observed. 

The blood-cells may exist singly and scattered, or may be grouped in 
large masses forming distinct clots or adherent to tube casts forming the so- 
called blood casts. In true renal hematuria the blood is intimately mixed 
with the urine, the individual corpuscles usually appearing as pale shadows 
or "ghosts." In hemorrhage from the bladder the urine may show the presence 
of blood-clots of irregular form and size. If the two-glass test be applied, 
the second glass will contain the more blood, while in hematuria of renal 
origin both glasses will show equal amounts. In some cases clots of blood 
in distinct casts are seen. In the chronic parenchymatous nephritis clots 
are rarely present, while in malignant disease of the kidney clots are relatively 
common. 

It is important in making a diagnosis from the presence of blood that 
extraneous sources of blood-cells be excluded. If the blood be of renal origin 
it will be associated with the presence of casts and epithelial cells while no 
such elements will be present from a hemorrhage lower down in the genito- 
urinary tract. Albumin will also be present in more or less amount. It 
has been stated that if the blood be derived from other than renal sources, 
the clear supernatant fluid in the centrifuge tube will be albumin-free. The 
writer has convinced himself that this is an error, as he practically always ob- 
tains faint albumin reactions in cases of hemorrhage other than renal. 

1 See Braasch, Jour. Am. Med. Assn., 1913, LXI, 936; Kemble, Wash. Med. Ann., 1914, 
XIII, 150; Kretschmer, Interstate Med. Tour., 1914, XXI, 1256; Wulff, Hospitalstid., 1914, 

lvh, 1465. 



356 



DIAGNOSTIC METHODS. 



(5). Casts. 

True casts are moulds of the uriniferous tubules. Their mode of forma- 
tion is not entirely clear. Undoubtedly a colloid substance is thrown into the 
lumen of the tubule and later solidifies forming a distinct cast of that particular 
tubule. In this process of hardening the material may enclose cells of different 
types which are, also, present in the tubule. Whether this coagulable materia] 
is derived from the blood as a transudate, whether it be a secretion of the 
epithelial cells which have become pathologic, or whether it be material arising 
from degeneration of renal cells is not at present settled, the latter source being 
the more probable. 1 In the urine we find true casts of the renal tubules, as 
well as pseudo casts which have nothing in common with the true type of 
these pathologic formations. 

True Casts. 
Hyaline Casts. 

The true hyaline casts are pale, transparent, homogeneous cylinders with 
rounded ends. Their size may vary from a very small fragment to one several 




Fig. 107. — Hyaline casts. One cast is impregnated with four renal cells. {Hawk.) 

mm. in length. In diameter they may be narrow or broad. As a rule, little 
difference is clinically made between these various types of hyaline casts, 
but the broader types seem to the writer to be somewhat more significant 
than do the narrower ones. The characteristics of the true cast are their 
cylindrical appearance, their sides being parallel and usually straight, although 
they may at times be observed in typical tortuous forms. They are never 
tapering at the ends, but may show an irregular outline at one or both ends, 
but the length and the parallel sides will usually differentiate them. 
1 See Erdman, Jour. Am. Med. Assn., 1912, LIX, 1952. 



THE URINE. 357 

The pure hyaline casts are perfectly homogeneous and free from granules. 
Such types are, however, not frequently observed as very fine granules may al- 
most always be detected embedded in the surrounding homogeneous materialt 
There may be even inclusions of epithelial, renal, blood-, or pus-cells, so tha. 
the gradations between the pure type of hyaline casts and many of the other 
varieties are outlined with difficulty. It should be stated at this point that 
a distinction exists between hyaline casts with enclosures of cells to such an 
extent that the cast is named from the variety of cell present and the type of 
pseudo cast in which groups of such cells are massed so as to form an apparent 
cast, but which do not have any definite matrix. The true hyaline cast is 
soluble in acetic acid and may be stained yellow with Lugol's solution. 

These hyaline casts are not always easy to find in the sediment. In 
examining the urine for the presence of casts the light should be shut off as 
much as possible and a low-power lens used. With the use of the high power 
the field is much limited and one is not so apt to observe the cast as with the 
low power. It is, however, always advisable to examine a cast, first seen with 
the low power, under the high power, so that the decision may be much more 
definite as to whether the cellular elements are real inclusions or simply material 
resting upon the true cast. The same is to be said regarding the presence 
of granules in the hyaline cast. 

Significance. 

Regarding the significance of hyaline casts in the urine, it is to be said 
that they occur in any condition in which the kidney is altered by circulatory, 
toxic, or inflammatory disturbances. They are not pathognomonic of any 
one condition and may be found as a result of simple functional disturbance. 
A few hyaline casts may be found in practically every urine, providing suffi- 
cient search is made. Any undue strain, such as running for a car in one 
who is not used to such exertion, may be sufficient to add quite a number 
of hyaline casts to the urine. In many thousand urine examinations made 
in the writer's laboratory, it has been rather the unusual thing not to find an 
occasional hyaline cast. It would seem, therefore, that no significance what- 
ever should be attached to the presence of an occasional hyaline cast. When, 
however, these casts become very numerous they should then be interpreted 
as meaning a disturbance of the kidney, although the absence of other types 
would rather speak against a marked pathologic change. In diabetes mellitus, 
" showers " of casts are especially observed preceding the appearance of coma 
(Kiilz). 

Granular Casts. 

These are modifications of the true hyaline cast in the sense that fine 
or coarse granules are found in the matrix of the hyaline cast. Several types 
of granular casts are observed. The granules may be very fine, very coarse, 
or may be distinctly composed of degenerated epithelial cells. The fine as 
well as the coarse granules are undoubtedly derived from the renal epithelium, 
which has degenerated completely. The coarser the granules the more severe 
the inflammatory process. 



358 



DIAGNOSTIC METHODS. 



These granular casts vary in shape and in size, but are usually shorter 
than the hyaline type. To these granular casts may be attached various cells 
so that it is difficult to tell whether the cast is really a true granular or a cellular 
one. In some cases these cellular inclusions may undergo fatty degenera- 
tion giving a much higher refractility to the specimen. Not infrequently one 
observes hyaline casts which are distinctly granular in one portion while 
the other is perfectly homogeneous. 

The so-called brown granular casts appear to be almost entirely degener- 
ated epithelial cells, although the coloring matter is probably hemoglobin. 




Fig. 108. — Granular casts, X 225. {Tyson.) 

The fact that the hyaline matrix cannot be distinctly made out does not 
argue against this type being truly hyaline in character, although the matrix 
is completely saturated with the pigment. 

Waxy Casts. 

This type is very refractile, transparent, and either perfectly colorless or 
showing a slight shade of yellow. Usually they are very long and broad and 
may be either straight or curved. The ends show a very distinct fracture 
while the cast itself may show a tendency to split transversely. Their ap- 
pearance is, therefore, that of ordinary wax. They may have any type of 



PLATE XI. 



a t 

II 



\ 



I 









Waxy Casts Treated with Iodixe. {Tyson) 



THE URINE. 



359 



cellular element attached and may show marked fatty degeneration. Some 
of these casts show the amyloid reaction while many of them do not. 

Waxy casts were at one time believed to be pathognomonic of amyloid de- 
generation of the kidney. It is true that they do appear earlier in this type of 
kidney lesion, but it is to be remembered that they occur in all varieties of 
chronic kidney disease. They are usually of bad prognostic omen as they in- 
dicate a very advanced process. 

Fibrinous Casts. 

These are very highly refractile, transparent, and always of a yellowish 
or brown color. They may be granular and have various cellular inclusions. 
Their shapes vary as do those of the hyaline types and they show a tendency 
to become fractured, the fracture usually being ragged, 
while in the waxy cast it is sharp-cut. 

Fibrinous casts usually appear in the acute renal 
conditions and disappear when this condition clears 
up. They do not have, therefore, the grave signifi- 
cance of the waxy cast and should be sharply differen- 
tiated. These casts are not composed of fibrin as their 
name would indicate, but are so called on account of 
their brownish color, which resembles fibrin. 

Epithelial Casts. 

These casts are true hyaline casts which include 
so many renal epithelial cells that the hyaline matrix 
may be lost. For the name epithelial cast to be ac- 
curate it is not necessary that more than a few cells 
be present. The cells may be well preserved or show 
marked fatty or granular degeneration. The nuclei 
of these cells are round and vesicular so that they may be easily recognized. 

Distinct gradations exist between the true epithelial cast and the coarsely 
granular and fatty cast. This type of cast is indicative of a severe destructive 
lesion of the kidney epithelium. 

Fatty Casts. 

These casts are masses of epithelial cells which have so markedly degener- 
ated that little is recognized beyond the original outline of the cell and the 
numerous fatty globules contained therein. They may be yellowish or black 
in color, the globules being soluble in ether and staining black with osmic acid 
or red with Sudan III. 

Blood Casts. 

These are casts including large numbers of blood-cells. The casts 
are formed within the tubules of the kidney, the cells occasionally being very 
pale. These casts indicate a serious advanced lesion of the renal parenchyma. 

Pus Casts. 

These like the other types of casts are true hyaline casts with enclosures 
of pus-cells. They are formed within the tubules of the kidney and usually 




Fig. 109. — Epithelial 
casts. (Hawk.) 



360 



DIAGNOSTIC METHODS. 



indicate an acute pyelonephritis. For the differentiation of these casts from 
epithelial casts it is advisable to add acetic acid to the sediment when the 
typical polymorphous character of the nucleus will distinguish the pus-cell 
from the epithelial cell with its vesicular nucleus. Moreover, the pus-cell is 
much more spherical than is the epithelial cell. 

Cylindroids. 

It is not infrequent to find in the urine formations which resemble the 
true hyaline casts to a marked degree. They, however, differ in the fact that 
at one or at both ends they taper off into a point which may be prolonged into 
a distinct thread. If, however, these ends are broken off, as may occur in 
the centrifugation, it is a practical impossibility to distinguish them from a 
hyaline cast. They are both found in the urine under the same conditions 
and their significance is practically the same. From the chemical standpoint 




Fig. no. — Fatty casts. {Hawk after Peyer.) 

they appear similar to the hyaline casts, their origin, therefore, being presum- 
ably in the renal parenchyma. If these bodies are true mucin and are insolu- 
ble in acetic acid, their origin is more probably in the bladder. 

A second type of cylindroid appears in the urine in the form of long tapering 
transparent shreds. They very much resemble ribbon which varies in diameter 
and may show under high power a distinctly fibrillar structure. These threads 
largely compose the nubecula. They are much longer than the hyaline cast 
and considerably narrower so that confusion should not arise. In cases of 
gonorrhea one finds mucous shreds which may vary from a few mm. to 1 cm. 
in length and yellowish or pure white in color. In the meshes of these shreds 
one finds embedded large numbers of pus and epithelial cells. These should 
be sharply differentiated from the true cast by their larger size and typical 



PLATE XII. 




Mucous Threads in Urine. (Unstained Specimen ) 



THE URINE. 



361 



mucoid character. Frequently they may be observed by the naked eye in 
large numbers. 

Pseudocasts. 

Not infrequently do we find in urine crystalline material arranged in 
masses much resembling casts. The most important of these are uric acid 
and the urates. It is true that any cast in a concentrated urine may become 
covered with urates so that the true nature of the cast becomes indefinite. 
If the slide be warmed these pseudourate casts will disappear, while the true 
casts will remain. Masses of bacteria, pus-cells, epithelial cells and blood- 
cells may so group themselves as closely to resemble true casts. As a rule 
such masses will show irregular outlines and no evidence of a distinct matrix. 
Moreover, the use of an old slide upon which there may be many scratches 




Fig. hi.— Blood, Pus, Hyaline and Epithelial Casts. {Greene.) 

a, Blood casts; b, pus cast; c, hyaline cast impregnated with renal cells; 

d, epithelial casts. 

should be avoided as the writer has seen several instances in which supposed 
casts were found to be due to such scratches. 

Cylindruria. 

This is the name given to the appearance of casts in the urine. As a rule, 
it should be said that the presence of a few hyaline casts is not of particular 
moment unless associated' with other evidences of marked renal disturbances. 
While albuminuria and cylindruria usually go hand in hand, yet we do find 
cases in which one exists without the other. 

It is undoubtedly true that casts indicate a disturbance of the renal epithe- 
lium. This, however, need not be anything more than disturbed nutritional 
or circulatory conditions. However, when the true hyaline casts are present in 



362 



DIAGNOSTIC METHODS. 



large numbers and when many other types of casts also exist, then a distinct patho- 
logic lesion of the kidney must be assumed. l As a rule, the granular types of cast 
are observed in the chronic processes, while the cellular forms are more usually 
present in the acute conditions. This rule, however, is not invariable, so that 
it may generally be stated that no type of cast is pathognomonic of any single 
condition. In this connection we should remember that recent work, especially 
that of Cabot, has shown that it is unwise to base a diagnosis of a kidney lesion 
upon the finding even of both albuminuria and cylindruria. So much discrep- 
ancy was shown to exist between the urinary and autopsy findings that one 
must remain in doubt as to whether it is possible to make a definite diagnosis 
unless clinical symptoms other than urinary are made the basis of a diagnosis. 
On the other hand, some of the most typical cases of nephritis, as shown post- 




Fig. 112. — Cylindroids. (Hawk after Peyer.) 

mortem, gave absolutely no indication in the urine that such condition existed. 
We are, therefore, face to face with the proposition that urinary examination 
must be in any case simply one of the diagnostic links. This fact is of special 
importance in life insurance examinations, as most companies absolutely 
refuse insurance to one who has ever shown albumin or casts in the urine. 
This would seem to the writer not only very short sighted, but based upon an 
absolutely erroneous idea of the importance of albumin and casts in the urine 
of one who showed absolutely no clinical signs of renal involvement. Remem- 
bering that albumin and casts may not appear, even though the kidney be 
seriously affected, it would seem just as plausible to refuse life insurance because 
these substances were not present. In this connection the writer would say 
that only when the urine is considered as a whole may definite conclusions be 

1 See Barringer and Warren, Arch. Int. Med., 1912, IX, 657; also, Posner, Berl. klin. 
Wchnschr., 1913, L, 2040; Minerbi, Policlinico, 1914, XXI, 1667. 



THE URINE. 363 

made regarding any type of renal disease. The clinician, who is thoroughly 
familiar with the course of the case, is the only one capable of interpreting the 
findings of the laboratory, so that it should be an unvarying rule for a laboratory 
worker to avoid diagnostic remarks unless he is thoroughly en rapport with the 
patient. The writer does not wish to be interpreted as stating that a diagnosis 
of renal disease may never be made from an examination of the urine, but he 
wishes to impress upon his readers that both albuminuria and cylindruria may 
occur without direct kidney disease or may not appear when such is present. 

(6). Spermatozoa. 

Spermatozoa are frequently observed in the urine of healthy adults, 
especially after intercourse or nocturnal emissions. In females they may 
also be observed as an evidence of intercourse, which fact is of some importance 
in cases of suspected rape. 

Pathologically, they may be found in cases of marked constipation, 
when the pressure of the impacted feces upon the seminal vesicles may induce 
an emission. In occasional cases of cystitis, associated with stricture, these 
bodies may be observed as reported by Simon. In cases of epilepsy and 
hysteroepilepsy, as well as in spinal disease following vertebral fractures and 
dislocations, spermatozoa are not infrequent. Masturbation and venereal 
excess frequently lead to almost constant spermatorrhea. Their occurrence in 
cases of prostatitis will be discussed in the section on Semen. 

(7). Tissue Fragments. 

It is not infrequent to find shreds of tissue in the urine, which may throw 
some light upon a pathologic condition. In cases of carcinoma of the bladder 
more rarely of the kidney, true malignant tissue may be obtained, which may 
permit of a tentative diagnosis, although the material is usually too necrotic 
to make an absolute diagnosis possible. 

(8). Bacteria. 
It should be stated in the beginning of this discussion that an examination 
of the urine for bacteria should be made only upon specimens obtained with 
the greatest possible precaution and preserved in absolutely sterile vessels. 1 
Soon after the urine is voided, especially if it remains in contact with the air, 
large numbers of saprophytic organisms may be found which, of course, did 
not exist in the original urine. In obtaining a specimen from the male it is 
not always necessary to catheterize the patient. If the surface of the glans 
and the orifice of the meatus be carefully washed with bichlorid solution 
followed by sterile water and the first portion of the urine voided be thrown 
away, the last portion may be collected in a sterile vessel and later put in work. 
With female patients, however, it is absolutely essential that catheterization 
be performed. The external genitalia and especially the orifice of the urethra 
are well washed with green soap and water. The opening of the urethra is 
then dried with sterilized cotton pads which are soaked in boracic acid. A 

1 See Hort, Jour. Hyg., 1914, XIV, 509. 



364 DIAGNOSTIC METHODS. 

sterilized glass catheter, whose external end is covered with a rubber tube 
about four inches long and large enough to fit loosely over the catheter is 
then inserted, care being taken that it touches only the orifice of the urethra. 
The urine is allowed to flow freely for a short time when the last portion is 
collected in a sterile vessel, the rubber tube being previously removed, 
(Kelly). Cultures are then made from the urine and the remainder centri- 
fuged in a sterile closed tube in order to throw down any bacteria which may 
be present. It is sometimes advisable, in order to diminish the specific gravity 
of the specimen, to add an equal volume of 95 per cent, alcohol and centrifuge 
the mixture. Practically all of the bacteria present will then be found in the 
sediment. 

The supernatant fluid is then removed by quickly inverting the tube 
and allowing the fluid to run out. The sediment, by this manipulation, will 
usually remain in the smaller portion of the tube. Smears are then made upon 
a glass slide and dried first in the air and then over the flame. It is not always 
the simplest matter to prepare smears which will remain after treatment with 
the staining solution, as the urea and salts of the sediment may be removed 
by the washing and carry with them the bacteria. If pus-cells are present, 
satisfactory smears are usually obtained; but if such conditions do not exist 
it is advisable to add a solution of egg albumin to the sediment before drying 
over the flame. 

The methods of staining the sediment for the various bacteria will depend 
entirely upon the organism supposed to be present. As a rule, a preliminary 
examination is very satisfactorily made by treatment with Lbffler's methylene- 
blue solution, which stains practically all organisms. If the tubercle bacillus 
is suspected it may be detected in exactly the same way as outlined under 
Sputum by staining with carbol-fuchsin solution. Should one suspect the 
presence of the gonococcus 1 this may be stained, as described in the next section, 
by Gram's method. Outside of these two types of organisms, it is almost 
impossible to differentiate the bacteria of the urine by staining methods. 

If the urine be collected with the precautions mentioned above, any organ- 
isms found must be attributed to their presence in the urine as voided. In 
this connection we must remember that the presence of the tubercle bacillus 
does not necessarily indicate tuberculosis along the genitourinary tract. 2 Tubercle 
bacilli are found in the urine in cases of miliary tuberculosis and have been re- 
ported in pulmonary tuberculosis, although it is more frequent to find them 
as evidences of local tubercular conditions. Of course, if large numbers of pus- 
and blood-cells be present along with tubercle bacilli, the diagnosis is usually 
certain. A word of caution is, however, necessary at this point. The smegma 
bacillus grows in abundance on the external genital organs and its morphological 
and staining characteristics may closely resemble those of the tubercle bacillus. 3 

1 This must not be confused with the micrococcus catarrhalis, which not infrequently is 
the etiologic factor in genitourinary inflammations. 

2 See Kielheuthues, Folia urol., 1912, VII, 191. 

3 See Gautier, Jour. d'Urol., 1914, V, 161; Brereton and Smith, Am. Jour. Med., Sc, 1914, 
CXLVIII, 267; Churchman, Ibid., 722; Editorial, Jour. Am. Med. Assn., 1915, LXIV, 348; 
Brown, Ibid., 886. 



THE URINE. 365 

If the proper precautions be observed, as they should be, no differentiation is 
necessary, bacilli showing the true morphological and staining character of 
the tubercle bacillus can be only this organism, as extraneous bacteria have 
been avoided. In the clinical laboratory, however, one may never be sure 
whether the proper precautions have been taken, so that absolute methods of 
differentiation (see Sputum) should be part of the technic unless the worker 
absolutely knows that contamination was avoided. A second point regarding 
the tubercle bacillus is that it may not be found by microscopic examination 
even after repeated attempts. Under such conditions the wisest»course to pur- 
sue is the inoculation of a guinea-pig with the washed urinary sediment. The 
obtaining of the urine must in this case be absolutely accurately done by observ- 
ing every precaution to prevent contamination. The reason for this is not 
because contaminating organisms will cause lesions similar to those of the 
tubercle bacillus, but because such secondary invaders may so infect the animal 
that death results from causes other than those for which we are looking. The 
washed sediment is injected intraperitoneally and the animal kept under ob- 
servation for three weeks to one month unless death results previously. At 
the end of this time the animal is killed and a postmortem examination made 
for evidences of tuberculosis. The retroperitoneal glands, spleen, and liver 
are the especial organs to show such lesions. These organs should be sectioned, 
portions run through the regular pathologic routine, and sections examined 
microscopically. A finding of tuberculosis in this way is unequivocal and is 
the quickest way in the long run of making a positive diagnosis, although a 
single examination may show the presence of tubercle bacilli in the urine, but 
rarely such is the case. 1 

Having found the tubercle bacilli in the urine, we are confronted with the 
question of the part affected. As a primary tuberculosis of the bladder is rare 
we may usually assume the seat of the difficulty to be the kidney, although if 
evidences of cystitis be present a combination may exist. As the symptoms of 
genitourinary tuberculosis are frequently vesical in origin, a kidney lesion 
may not be suspected, but should be assumed until the contrary is proven. 
Thanks to the introduction of methods of cystoscopic examination and espe- 
cially ureteral catheterization, we are in a better position to make a positive 
diagnosis of renal tuberculosis and exclude that of bladder origin. An inter- 
esting point regarding tuberculous cystitis is that the urine, although frequently 
containing large amounts of pus, is practically always acid in reaction. More- 
over, this pus is frequently sterile in tubercular cystitis. It is not the province of 
the writer, at the present time, to outline methods of differential diagnosis of 
various conditions; he will refer, therefore, to works on genitourinary diseases 
for the various types of cystitis and their clinical differentiation. Suffice it to 
say at this point that cystitis may be an ascending or a descending one and should 
always be correlated with the associated condition. It is usually a simple 
matter to determine the presence of the gonococcus in the urethral discharge, 
but it is far from easy to demonstrate this organism in the case of gonorrheal 

1 See Bryan, New York Med. Jour., 1913, XCVIII, 20; also, Keene and Laird, Am. Jour. 
Med. Sc, 1913, CXLVI, 352. 



366 



DIAGNOSTIC METHODS. 



cystitis. Under such conditions the symptoms and the association with an 
existing gonorrhea would furnish the decisive clue. 

Bacilluria. 

By this condition is meant the presence in the freshly voided urine of so 
many organisms that the urine is distinctly cloudy. These organisms are usu- 
ally those associated with a mild cystitis or may be those of an existing general 
infection. 1 In cases of persistent bacilluria, we may find a true renal origin, 
which is largely associated with the presence of the typhoid and colon bacillus. 
As has been well established, the typhoid organism may be excreted for months 
after the patient is convalescenent so that it becomes necessary to use strict 
measures to disinfect all urine of typhoid patients. The colon bacillus is at 

present assuming so much importance in 
clinical work that cases are being almost 
daily recognized which may be directly 
traceable to the colon bacillus and not to 
the typhoid as usually assumed. It 




is 



•*> \ 



necessary, therefore, for the laboratory 
worker especially, and, where possible, for 
the general practitioner to be able to recog- 
nize each of these organisms when present 
either in the feces, urine, milk, or water- 
supply (see Feces). A second general class 
of cases associated with bacilluria are those 
of urethritis and prostatitis. Usually there 
is a secondary cystitis arising from the same 
organism or, at least, the resistance of the 

bladder has been so far reduced that these organisms which find their way 

into the bladder develop profusely therein. 



tfatMnne M ill 

Fig. 113. — Scolex and hooklets of 
taenia echinococcus in urine. 



(9). Parasites. 
Various types of parasites are observed in the urine. Thus the tricho- 
monas vaginalis has been found by Kiinstler, Miura, and Dock. Amebae 
have been found by Balz, Jurgens, Wijchoff, and by Musgrave and Clegg. 
Various portions of hydatid cysts are frequently observed, among which we 
find the echinococcus hooklets and fragments of membrane. Nematode 
worms, especially the filaria sanguinis hominis, are present in cases of 
chyluria, while the anguillula aceti or " vinegar-eel' ' has been reported, 
especially by Stiles, while Billings and Miller report its presence as a possible 
contamination from the bottle in which the urine was collected. 2 

1 Freifeld (Berl. klin. Wchnschr., 1913, L, 1761) reports the finding of diphtheria bacilli 
in urine. Rosenow (Jour. Infect. Dis., 1909, VI, 296) and Townsend (Jour. Am. Med. Assn., 
1913, LXI, 1605) report the pseudo-diphtheria bacillus as a causative factor of cystitis. See, also, 
Curtis, Jour. Am. Med. Assn., 1915, LXIV, 270; Surg. Gyn. and Obs., 1915, XXI, 423. Barber 
and Draper, Jour. Am. Med. Assn., 1915, LXTV, 205; McGowan, Ibid., 226; Cunningham, 
Ibid., 231; Koll, Ibid., 297; Greeley, New York Med. Jour., 1915, CI, 250; Hoover, Interstate 
Med. Jour., 1915, XXII, 163; Kretschmer, Ibid., 173. 

2 King (Jour. Am. Med. Assn., 1914, LXIII, 2285) reports a case of Myiasis of the urinary 
passage due to the presence of the larva of the small "latrine fly" (fannia scalaris). 



PLATE XIII. 



t 

m 









Cystitis due to Colon Bacillus. (Methylene Blue Stain.) 



PLATE XIV 



zgm ? 



» • 



step 



# 



Staphylococcus Cystitis. (Leishman Stain.) 



THE URINE. 



367 



Eggs of the schistosomum haematobium are not infrequently observed 
together with large numbers of red cells in cases of bilharziasis. This worm 
as well as its ova will be discussed in the section on Blood. Stuertz has 
reported the findings of the egg of eustrongylus gigas in the urine in a case 
of chyluria. 

V. Calculi. 

Concretions of a more or less hard and dense character are prone to form 
in the urinary passages. These bodies are termed, according to their size 
and location, sand, gravel, stone, and calculi. These formations consist of 
accretions of any of the various crystalline or amorphous sediments previously 
mentioned, the type of stone depending upon the reaction of the urine. 

These calculi are formed by the deposition of the crystalline material 
around a definite nucleus, which usually consists of organic material, such as 
fibrin, blood, desquamated epithelial cells, mucus, or even a crystal of uric 
acid or calcium oxalate. 1 It is very difficult to decide as to the reason for the 
deposition of this material in the form of a renal stone. The growth of the 






Fig. 114. — Ova and miracidium of schistosomum hematobium, X 300: A, Ovum as seen 
in urine; B, the same after addition of water; C, miracidium. {Tyson after Raittiet.) 

calculus takes place by accretion, the deposition of successive layers of material 
occurring around the original nucleus. The material of which the stone con- 
sists will usually be of one kind, so that we speak of uric acid or phosphate 
calculi, for instance, while, occasionally, mixed calculi may be formed by the 
deposition of two or more chemical combinations. 

The classification of urinary concretions is based on the chemical constituents 
of which they are composed. Before examining a calculus chemically a thorough 
optical examination should be made, as this may give a definite clue as to its 

iSeePosner, Ztschr. f. Urol., 1913, VII, 799; also, Lichtwitz, Ibid., 810; also, Williams, 
New York Med. Jour., 1915, CII, 609. 



368 DIAGNOSTIC METHODS. 

composition. After this preliminary examination, the calculus is ground to 
a fine powder and examined according to the following table of Heller. 

Uric Acid Calculi. 

These are, perhaps, the most common renal stones. They are not always 
composed of pure uric acid, but are made up of a mixture of this substance 
with the urates. They are always colored, usually yellowish or brownish, 
but may at times appear distinctly red. They are fairly hard and usually 
show a rough irregular nodular surface, although at times this may be smooth. 
They fracture very easily and show, on cross section, a distinctly laminated 
structure, the layers frequently being of different colors, in some cases 
even being composed of deposits other than uric acid. If heated on a plati- 
num foil they are combustible, burning without a flame. They give the murexid 
test and do not liberate appreciable amounts of ammonia on treatment with 
sodium hydrate. 

Ammonium Urate Stones. 

The ammonium urate calculi occur rarely in the adult. They are small, 
yellow and very soft, being distinctly clay-like and easily powdered when 
dry. These stones give the murexid test and also give a strong reaction 
for ammonia on treatment with sodium hydrate. 

Calcium Oxalate Stones. 

Next to the uric acid calculus the oxalate stone is most frequently met. 1 
The smaller types of these calculi are practically colorless and have a smooth 
surface, while the larger ones are grayish, brownish or even black in color 
and have a rough nodular surface with sharp projecting angles. These 
stones frequently cause severe hemorrhage and much irritation when passing 
through the ureter and urethra. From their appearance they have been called 
the mulberry calculi. They are, perhaps, the hardest of the urinary stones. 

These calculi are insoluble in acetic acid, but soluble in hydrochloric 
acid without effervescence unless the powder is previously heated. It is rare 
to find these stones perfectly pure, admixtures with various other sediments 
leading to distinct concentric arrangement as shown on fracture. 

Phosphatic Calculi. 

Stones composed of pure alkaline phosphates or triple phosphates are 
exceedingly rare. Usually the phosphatic calculi contain admixtures of 
ammonium urate, calcium carbonate, and calcium oxalate. The color of such 
stones may range from a white through yellow to some with distinct reddish 
tones. They are frequently of very large size, especially when formed in the 
bladder, are of a chalky consistency, and show a rough surface. 

These calculi are soluble in hydrochloric or acetic acid, such solutions 
giving reactions both for phosphoric acid and the alkaline earths. 

1 See Rowlands, Biochem. Jour., 1908, III, 346; Kahn and Rosenbloom, Jour. Am. Med. 
Assn., 1912, LIX, 2252; also, Kahn, Arch. Int. Med., 1913, XI, 92; Kahn, Ztschr. f. exp. Path, 
u. Therap., 1915, XVII, 88; Rosenbloom, Jour. Am. Med. Assn., 1915, LXV, 161; Cabot. 
Surg. Gyn. and Obs., 1915, XXI, 403; Braasch and Moore, Jour. Am. Med. Assn., 1915, 
LXV, 1234. 



THE URINE. 



369 














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370 DIAGNOSTIC METHODS. 

Calcium Carbonate Calculi. 

Such stones are exceedingly rare. They are small in size, are distinctly 
chalk-like in consistency and color, and have a smooth surface. If treated 
with acid, carbon dioxid is evolved. 

Cystin Calculi. 

These stones are white or pale yellow in color, have either a smooth 
or irregular surface, and are soft and waxy in consistency. They vary in 
size occasionally being found as large as a hen's egg, although those of true 
renal origin are about the size of a pea. The formation of such calculi and 
their passage through the ureter and urethra constitute practically all of the 
untoward symptoms shown by subjects affected with cystinuria. 1 

Such stones burn readily if heated on a platinum foil, giving off a peculiar 
sharp odor. The powder is soluble in ammonia from which the characteristic 
hexagonal plates separate on allowing the ammonia to evaporate. 

Xanthin Calculi. 

These stones occur especially in children, although even here they are very 
rare. They are usually light brown in color, moderately hard, and vary in 
size from that of a pea to a tennis-ball. On cross section they appear amorphous 
and if rubbed take a polish much resembling that of wax. The powder shows 
the typical reaction for xanthin previously outlined. 2 

Urostealith Calculi. 

These masses consist of fat, calcium and magnesium soaps, and choles- 
terin. They are usually soft and may be somewhat irregular in shape. This 
material burns with a pale yellow flame giving an odor of resin. The dry 
powder is soluble in alcohol and ether, from which rhombic notched plates of 
cholesterin separate on evaporation. 

VI. Functional Diagnosis. 

It is usually of great importance, especially in cases in which surgical 
intervention is contemplated, to know just exactly what the functional capa- 
bilities of the kidney are. If one kidney is to be removed, the question arises 
as to whether the remaining kidney can sufficiently accommodate itself toJ:he 
increased work which must be put upon it. 

As such a variation has been occasionally found between the results of the 
chemical and microscopical examination of the urine on the one hand and the 
pathologic condition shown in the kidneys postmortem on the other, an attempt 
has been made to find delicate tests by which a true renal lesion might be 
indubitably determined and thus permit of an absolute diagnosis even though 
the urinary findings were or were not conclusive. It is granted that a more 
or less severe lesion of the kidney may exist and yet its functional capacity 

1 See Frankenthal, Deutsch. Ztschr. f. Chir., 1914, CXXXI, 442; Neumann, Deutsch. med. 
Wchnschr., 1914, XL, 2065. 

2 See Rosenbloom, New York Med. Jour., 1915, CI, 120. 



THE URINE. 371 

be almost normal. If this functional capacity can be determined by tests 
which are more or less simple, it is evident that such methods should form part 
of the daily routine of the practitioner. 1 

Before discussing some of these tests, which have been advocated for 
estimating the functional activity of the kidney, the writer would say that few 
of them have proven entirely satisfactory. If we are to regard nephritis as a 
constitutional condition with secondary renal manifestations, it is self-evident 
that such tests can show only the manner in which a normal or pathologic kidney 
reacts toward certain substances and can have little reference to the primary 
etiologic factors. 

Cryoscopy. 

The method of determining the freezing-point of a solution is one of the 
most delicate of those of physical chemistry. As it is based upon the principle 
that substances in solution lower the freezing-point of the solvent in direct 
proportion to the molecular or ionic concentration of the solution, this method 
serves as a ready means of determining the molecular weight of a substance 
as well as the molecular concentration of a solution. For a successful outcome 
of a cryoscopic determination, the most assiduous attention to detail must be 
paid, so that this method certainly can find no place in the hands of the general 
practitioner or even in those of the laboratory worker, who has not been espe- 
cially trained along these lines. Such being the case, it is absurd to expect that 
slight variations in the freezing-point (A) of such a complex mixture as the 
urine can yield any valuable information, especially when one remembers that 
fluctuations wider than those shown under pathological Conditions may be noted 
as a result of not observing such slight details as that of constantly agitating the 
urine during the cooling and freezing. The normal freezing-point of the urine 
varies between — 0.9 and — 2 C, an increase being known as hypersthenuria 
and a decrease as hyposthenuria. 

As will be seen in the discussion of this subject in the section on Blood, 
the results obtained by this method have been far from satisfactory, as nothing 
of distinct diagnostic value has as yet been derived from comparative studies 
by various workers. It would seem to the writer, therefore, that for the 
present this method would better be left to the research worker than to be adopted 
by the general or special student, who should make use of methods which will 
yield results of more immediate value to Jaim. For these reasons the writer 
must refer to other works for a detailed description of the method. 

Electric Conductivity. 

The remarks made later in the section on Blood regarding electric conduc- 
tivity are especially applicable to the urine. This test is altogether too delicate 
to be applied to such complex fluids as the urine with the hope that slight 
variations in the conductivity will show anything of importance. As the 

1 See Blum, Renal Diagnosis in Medicine and Surgery, New York, 1914; Stevens, Jour. 
Am. Med. Assn., 1914, LXII, 1544; Geraghty, New York Med. Jour., 1914, C, 3i2;Fiersol, 
Interstate Med. Jour., 1914, XXI, 1165; Thomas, Jour. Am. Med. Assn., 1914, LXIII, 1909; 
Braasch and Thomas, Ibid., 1915, LXIV, 104; Geraghtv, Bull. Johns Hopkins Hosp., 1915, 
XXVT, 155. 



372 DIAGNOSTIC METHODS. 

electric current is conducted only by dissociable compounds, this method 
can show absolutely nothing regarding the excretion of the nondissociable 
organic substances. Such being the case the writer can see no reason for resort- 
ing to such delicate procedures as the determination of the conductivity of the 
urine when no attempt is made, as may be observed in many of the experiments 
reported, to control the intake of the inorganic substances which would affect 
the conductivity of the urine. A little more attention to ordinary methods 
of chemical examination, with especial regard to ascertaining the intake and 
output of the patient, would, in the writer's opinion, yield much more valuable 
information than could be obtained by the rather uncertain urinary manipu- 
lations with the method of Kohlrausch. 

Chlorid Excretion. . 

By the determination of the chlorid excretion in a given case under the 
influence of a specific intake of sodium chlorid it was hoped that some informa- 
tion might be derived as to the "glomerular sufficiency" of the kidney. This 
test, known as alitnentary chloruria, advanced by Claude and Mante, 1 has 
like the above yielded little of value. The exceptions to the general rule 
of pathologic retention are too numerous to permit of absolute differentiation. 
While it has been shown by Widal that a retention of sodium chlorid does 
occur in nephritis and that a restriction of the salt in the diet frequently does 
alleviate the edema and albuminuria, yet many factors not well understood fre- 
quently give rise to opposite results (see Chlorids in Urine). The general consen- 
sus of opinion regarding this test may be summed up by the statement that the 
chlorid excretion in cases of health and disease is of no distinct value in diag- 
nosing the functional activity of the kidney. 

Methylene Blue Test. 

It was hoped by the introduction of such tests that the capability of the 
kidney to excrete substances present in the blood could be determined by 
the ease with which artificially introduced substances were eliminated. Achard 
and Castaigne administer by the mouth o.i gram of methylene blue in a capsule 
or, preferably, 0.05 gram subcutaneously. This substance is excreted by the 
kidneys in the form of a colorless chromogen in 15 to 30 minutes after injection. 
This chromogen may be converted into the colored pigment by heating with 
acetic acid. In about five minutes after the excretion of the chromogen, the 
urine becomes greenish-blue from the excretion of the pigment itself. Under 
normal conditions the excretion reaches a maximum in from three to four hours 
and may last two to three days. If the pigment is not excreted within one hour 
after subcutaneous injection, pathologic conditions are supposed to exist. 

In some cases of chronic interstitial nephritis it was found that such 
injections were not followed by an excretion of the pigment until one to three 
hours had elapsed. This would be a valuable diagnostic method had it not 
also been shown conclusively that many cases of advanced interstitial nephritis 
show the same excretion of this pigment as normal individuals. Such being 
the case the test is of no more use than many clinical and routine urine methods. 
1 Arch. gen. de med., 1902, VTII, 129. 



THE URINE. 373 

Phloridzin Test. 

This test is based on the assumption that phloridzin normally gives rise to a 
glycosuria through distinct alternations in the renal cells. In other words, a 
so-called "renal diabetes" is set up. The technic is as follows: i c.c. of a 
1:200 aqueous solution of phloridzin is injected subcutaneously. The urine is 
tested at intervals of 15 minutes for the appearance of sugar. Normally, sugar 
may be detected in one-half to one hour and may be present for as long as five 
hours. The quantity eliminated may vary from 0.5- to 3 grams of glucose. In 
nephritis the sugar is usually absent or below 0.5 gram. This test does not 
distinguish the various types of nephritis, yet it does usually indicate that renal 
activity is disturbed. It is, however, being rapidly displaced by the following 
test. 

Phenolsulphonephthalein Test. 

Rowntree and Geraghty 1 have introduced a test for the functional activity 
of the kidney which is by far the most accurate at our disposal. 2 The solution 
used is prepared as follows: 0.6 gram of phenolsulphonephthalein and 0.84 c.c 
of 2/N NaOH solution (8 per cent.) are added to sufficient 0.75 per cent. NaCl 
solution to make 100 c.c. Each c.c. of this solution contains, therefore, 6 mg. 
of the dye. As the mono-sodium salt, formed in this solution, is slightly irritant 
locally when injected, 2 or 3 drops more of the 2/N NaOH are added, when the 
solution changes to a beautiful Bordeaux red color and becomes non-irritant. 3 

The technic is as follows: 20 to 30 minutes before applying the test, the 
patient is given 300 to 400 c.c. of water in order to insure a free urinary secretion, 
otherwise delayed time of appearance may be due to lack of secretion. Under 
aseptic precautions a catheter is introduced into the bladder and the bladder 
completely emptied. Noting the time, 1 c.c. of the above solution is administered 
subcutaneously in the upper arm by means of an accurately graduated syringe. 
The urine is allowed to drain through the catheter into a test-tube in which has 
been placed a drop of 25 per cent. NaOH solution and the time of the first pink- 
ish tinge is noted. In patients without urinary obstruction, the catheter is 
withdrawn at the time of appearance of the drug in the urine and the patient is 
instructed to void into a receptacle at the end of one hour and into a second 
receiver at the end of the second hour. A rough estimate of the time of ap- 
pearance may be made by having the patient void urine without the use of the 
catheter at frequent intervals. In prostate cases it is wise to have the catheter 
in place until the end of the observation. The catheter is corked at the time of 
appearance of the drug in the urine and the cork is removed at the end of the 
first and second hours, the bladder being drained each time. 

1 Jour. Pharm. and Exper. Therap., 1910, I, 579; Arch. Int. Med., 1912, IX, 284. 

2 See Conzen, Deutsch. Arch. f. klin. Med., 1912, CVIII, 353; Smith, Am. Jour. Dis. 
Child., 1913, V, 25; Pepper and Austin, Am. Jour. Med. Sc, 1913, CXLV, 254; also, Rown- 
tree, Fitz and Geraghty, Arch. Int. Med., 1913, XI, 121; Geraghty, Rowntree and Cary, Am. 
Surg., 1913, LVIII, 800; Dietsch, Ztschr. f. Exper. Pathol, u. Therap., 1913, XIV, 512; 
Rowntree, Geraghty and Marshall, Surg. Gynec. and Obs., 19 14, XVIII, 196. 

3 This mono-sodium salt of phenolsulphonephthalein may be obtained from Hynson, 
Wescott and Co. of Baltimore in the form of ampules, each c.c. of the solution containing 
6mg. 



374 DIAGNOSTIC METHODS. 

Each sample of urine is measured and the specific gravity taken. Suffi- 
cient 25 per cent. NaOH is added to make the urine decidedly alkaline in order 
to elicit the maximum color (a brilliant purple-red) . This solution is now placed 
in a liter volumetric flask and distilled water added to the 1 L. mark. Thoroughly 
mix the solution and filter a small portion for comparison with the standard 
solution. This standard consists of 3 mg. of phenolsulphonephthalein (or 
1 c.c. of the solution used for injection) diluted to 1 liter and made alkaline with 
1 or 2 drops of 25 per cent.- NaOH solution. This solution will retain its inten- 
sity of color for weeks. The comparison of color is made in the Duboscq or 
the Rowntree and Geraghty modification of the Autenrieth-Konigsberger 
colorimeter. The known (standard) solution is adjusted to the 10 mm. mark 
and the intensity of the unknown solution is made to correspond by means 
of the usual manipulations. One may readily calculate the amount of drug 
eliminated as follows: If, for instance, the reading for the unknown solution is 
20, it is evident that only 50 per cent, as much dye is present as is contained in 
the standard solution. That is, the excretion equals 50 per cent, of the 3 mg. of 
the standard or 25 per cent, of the 6 mg. injected. The readings may be then 
made on a second and a third voiding until the drug is completely eliminated. 

In normal cases it has been found that the time of appearance varied from 
5 to 1 1 minutes and that 40 to 60 per cent, of the drug was excreted in the first 
hour and 20 to 25 per cent, in the second hour. The excretion of the drug does 
not run parallel to the excretion of water. The smaller the amount of urine in 
normal cases the greater the concentration of the drug. It is immaterial, as 
far as the excretion of the drug is concerned, whether the urinary output is 
50,200,400 or more c.c. 

In pathological cases it has been demonstrated that the permeability of the 
kidney for this drug is decreased in both chronic parenchymatous and chronic 
interstitial nephritis, the decrease being most marked in the latter and varying 
with the intensity of the disease. In cases with obstruction in the lower urinary 
tract, this test may show renal involvement to such an extent that operation 
should be deferred, especially if the time of appearance is delayed beyond 25 
minutes and the output of the drug is below 20 per cent, for the first hour. 
This point is of special value to the surgeon as it shows the danger of using 
surgical intervention until the kidney may become less insufficient. It would 
seem to be of value, also, in the study of the renal condition preceding uremia. 
To the obstetrician it may show the possible approach of eclampsia, as has been 
demonstrated in several cases. In unilateral and bilateral kidney diseases the 
absolute amount of work done by each kidney as well as the relative proportion 
can be determined by resort to ureteral catheterization followed by this test. 
The writer has no hesitancy in advising a careful study of this test in all cases 
with suspected or actual renal involvement. 1 

1 See Behrenroth and Frank, Ztschr. f. exper. Path. u. Therap., 1913, XIII, 72; Rowntree 
and Fitz, Arch. Int. Med., 1913, XI, 258; Geraghty, Jour. Am. Med., Assn., 1913, LX, 191; 
Goodman, Ibid., 1913, LXI, 184; Frothingham, Fitz, Folin and Denis, Arch. Int. Med., 
1913, XII, 245; Geraghty and Rowntree, Jour. Am. Med. Assn., 1913, LXI, 939; Foster, 
Arch. Int. Med., 1913, XII, 452; Fishbein, Jour. Am. Med. Assn., 1913, LXI, 1368; Roth, 
Berl. klin. "Wchnschr., 1913, L, 1609; Marogna, Gazz. d. osp., 1914, XXXV, 172; "Widal, 
Weill and Radot, Presse med., 1914, XXII, 565; Pezzana, Policlinico, 1914, XXI, 563; Mach- 



THE URINE. 375 

BIBLIOGRAPHY. 

i. Benedict. Influence of Inanition on Metabolism. Washington, 1907. 

2. Blarez. L'Urine au point de vue clinique et medical. Paris, 1906. 

3. Blumenthal. Pathologie des Harnes am Krankenbett. Berlin, 1903; 

Handbuch der Speziellen Pathologie des Hams. Berlin, 19 13. 

4. Chittenden. Physiological Economy in Nutrition. New York, 1905. 

The Nutrition of Man. New York, 1907. 

5. Cohnheim. Chemie der Eiweisskorper. Braunschweig, 1906. 

6. Croft an. Clinical Urinology. Chicago, 1907. 

7. Daiber. Mikroskopie der Harn-Sedimente. Wiesbaden, 1906. 

8. Fischer. Untersuchungen ueber Aminosauren. Berlin, 1906. 

9. Gerard. Traite des Urines. Paris, 1906. 

10. Gerard. L'analyse des urines consideree comme un des elements de diag- 

nostic. 2* ed. Paris, 19 13. 

11. Hammarsten. Lehrbuch der physiologische Chemie. Wiesbaden, 1914. 

12. Heitzmann. Urinary Analysis and Diagnosis. New York, 1915. 

13. Hoppe-Seyler-Thierfelder. Handbuch der physiologisch- und path- 

ologisch-chemischen Analyse. Berlin, 1909. 

14. Krehl. Ueber die Stoning chemischer Korrelationen im Organismus. 

Leipzig, 1906. Pathologische Physiologie. Leipzig, 1912. 

15. Mann. The Physiology and Pathology of the Urine. London, 1913. 

16. Naunyn. Der Diabetes Mellitus. Wien, 1907. 

17. Neubauer und Huppert. Analyse des Harns. Wiesbaden, 1910 and 1913. 

18. Neuberg. Der Harn. Berlin, 191 1. 

19. von Noorden. Die Zuckerkrankheit. Berlin, 19 12. 

20. Ogden. Clinical Examination of the Urine. Philadelphia, 1903. 

21. Pavy. Carbohydrate Metabolism and Diabetes. London, 1906. 

22. Purdy. Practical Urinalysis and Urinary Diagnosis. Philadelphia, 1900. 

23. Rieder and Delephine. Urinary Sediments. London, 1899. 

24. Saxe. Examination of the Urine. Philadelphia, 1906. 

25. Scott. Clinical Examination of the Urine. Philadelphia, 1900. 

26. Spaeth. Untersuchung des Harns. Leipzig, 1903. 

27. Sutton. Volumetric Analysis. Philadelphia, 1904. 

28. Tyson. Bright's Disease and Diabetes. Philadelphia, 1904. 

29. Wells. Chemical Pathology. Philadelphia, 1914. 

witz, Rosenberg and Tschirtkoff, Munch, med. Wchnschr., 1914, LXI, 1268; Hess, Ibid., 
1835 and 1874; Ware, New York Med. Jour., 1914, XCIX, 416; Robertson, Ibid., 972; Jones, 
Ibid., 1914, C, 518; Fanz, Ibid., 1214; Tracy, Surg., Gyn. and Obs., 1914, XLX, 734; Block, 
Jour. Am. Med. Assn., 1914, LXII, 1309; Griessmann, Deutsch. Arch. f. klin. Med., 1914, 
CXTV, 32; Deutsch and Schmuckler, Ibid., 61; Hedinger and Schlayer, Ibid., 120; Hessel, 
Ibid., 396; Lichtwitz and Stromeyer, Ibid., 1914, CXYI, 127; Rowntree, Am. Jour. Med. Sc, 
1914, CXLVH, 352; Fitz, Ibid., 1914, CXLVIII, 330; Thayer and Snowden, Ibid., 781; Agnew, 
Arch. Int. Med., 1914, XIII, 485 ; Frothingham and Smillie, Ibid., 1914, XTV, 514; Quimby and 
Fitz, Ibid., 1915, XV, 303; Miller and Cabot, Ibid., 369; Rowntree, Marshall and Baetjer, 
Ibid., 543; Hopkins and Jones, Ibid., 964; Bauer and Habetin, Ztschr. f. Urol., 1914, VIII, 355; 
Bauer and Nyiri, Ibid., 191 5, IX, 81; Tileston and Comfort, Am. Jour. Dis. Child., 1915, X, 
278; Hess, Bull. Johns Hopkins Hosp., 1915, XXVT, 52; Smith, Jour. Am. Med. Assn., 1915, 
LXTV, 223; Elliott, Ibid., 1885; Pedersen, New York Med. Jour., 1915, CI, 770; Siegel, Pub. 
Jefferson Med. Coll., 1915, VI, 78. 



CHAPTER VII. 
SECRETIONS OF THE GENITAL ORGANS. 

I. Male Secretions. 

General Considerations. 

The normal secretion of the male generative organs is known as semen 
and is a mixture of the secretions of the prostate gland, the glands of Cowper, 
the testicles, and the seminal vesicles. It is a practical impossibility from the 
clinical standpoint to separate the different elements of the semen, so that this 
must be discussed as a whole. 

The semen is a white or slightly yellowish, somewhat thick and viscid 
liquid with a peculiar odor, somewhat resembling fresh glue, and showing a 
neutral or faintly alkaline reaction, a nonhomogeneous milky appearance, and 
a specific gravity greater than that of water. It is composed of semisolid 
material in the form of white masses floating in a limpid liquid and holding 
in suspension specific elements, derived from the secretory glands of the genital 
apparatus and from the desquamation of the various canals through which 
the semen passes. 

Semen contains about 6 per cent, of organic and 4 per cent, of inorganic 
matter. Its chief chemical characteristic is the presence of spermin (C 2 H 5 N) 2 , 
which is at least isomeric, if not identical, with diethylen-diamin, according 
to Ladenburg and Abel. This spermin, which is derived largely from the 
prostate gland, combines with the phosphoric acid radical to form spermin 
phosphate, which crystallizes in the form of four-sided spindles or prisms 
which may appear as flattened needles. In some cases these crystals resemble 
very closely the diamond-shaped double pyramids known as Charcot-Leyden 
crystals, which are found in the sputum. They are, however, of a different 
crystalline group and are soluble in formalin, while those found in the sputum 
are insoluble in this menstruum. These spermin crystals are known as Bott- 
cher's crystals. Miescher has studied the composition of the heads of the 
spermatozoa and has been able to isolate certain bodies, known as protamins, 
which are the simplest type of protein material. 1 

Microscopic Examination. 

The most important and characteristic constituent of semen are the sper- 
matozoa. 2 These sexual elements consist of an anterior broader portion or head 
and a narrow thread-like tail. The former is oval or egg-shaped and meas- 
ures about 5 microns in length, 4 in breadth, and 2 in thickness. Just behind 

1 See Steudel, Ztschr. f. physiol. Chem., 1913, LXXXIII, 72. 

2 Koessler (Trans. Chic, Path. Soc, 19 12, VIII, 280) has shown that the spermatozoa may 
act as direct carriers of infectious agents, among these being the spirochaeta pallida. 

376 



SECRETIONS OF THE GENITAL ORGANS. 377 

this pyriform head is a short cylindrical portion measuring 6 microns in length, 
which is known as the middle piece. This tapers somewhat to the point of 
union with the tail. This so-called tail is a thread-like posterior portion and 
is approximately 45 microns in length. In the freshly voided semen these tail 
portions show active undulatory whip-like motions, which persist for 24 to 
48 hours, and even longer under proper conditions, and enable the spermatozoa 
to progress from point to point. Alkalies seem to favor this movement, while 
dilute acids inhibit it very rapidly. This movement of the spermatozoa is 
closely associated with their sexual activity, as the cells showing no movement 



XaThcvnne. Hill. 
Fig. 115. — Normal semen. 

when freshly voided may be regarded as possessing no functional power. 
For a discussion of spermatogenesis as well as of fertilization of the ovum 
the writer would refer to the admirable description of McMurrich. 1 

Besides these characteristic portions of the semen, large numbers of 
lecithin globules are seen, which give the milky appearance to the fluid. Espe- 
cially to be noted among the cellular elements are the so-called corpora amylacea 
which resemble very closely starch granules, having concentric striations, a 
finely granular center and occasionally a nucleus. These cells take a distinct 
blue color on treatment with iodin solution. Moreover, various epithelial 
cells are observed, which are derived from the several glands contributing to the 
composition of the semen. Some of these cells are distinctly granular, some 
1 The Development of the Human Body, Philadelphia, 1907. 



378 DIAGNOSTIC METHODS. 

contain fat globules and very closely resemble the colostrum corpuscles of the 
lacteal secretion, while some of the granules resemble myelin. In rare cases 
cylindrical casts are seen which simulate the hyaline casts of the urine, but 
as a rule they are larger and longer. These are supposed to be derived from 
the prostate gland and seminal vesicles. If the semen be allowed to stand for 
a few minutes, several types of crystal may be observed, especially the phosphate 
of spermin, ammonium-magnesium phosphate, fatty acids, and oxalate of 
calcium. This last crystalline component is especially noted in the urine in 
cases of spermatorrhea. 

Pathologic Variations. 

Direct pathologic variations in the semen are limited to two conditions. 
Either spermatozoa are absent or those present are nonmotile. The deter- 
mination of the presence of spermatozoa in the semen or in suspected stains is 
a matter of simple microscopic examination. With the question of the motility 
of such elements, when present, the conditions under which the examination is 
made may markedly influence the findings. If possible, semen should be 
examined, with regard to the motility of the spermatozoa, as soon as ejected, 
but if such is not possible the fluid must be kept warm until examination may 
be made. It is absolutely out of the question to make a positive diagnosis 
of true nonmotility of spermatozoa from examination of specimens, which have 
been allowed to cool. In some cases, if the time has not been too extended, 
warming may bring back the motile power of these cells, but in many cases 
it does not. It is, therefore, unjust and unwise to brand a man as sterile 
without absolute proof that such a condition really exists. If no spermatozoa 
are present, especially after several examinations, sterility is absolute. This 
condition is known as azoospermatism. According to Kehrer, 40 per cent, of 
cases of conjugal sterility are due to the absence of spermatozoa in the semen. 
It is, therefore, necessary that the ordinary gynecological idea that women are 
the responsible factors in the family sterility should be, at least, partly borne by 
the man, as it is unjust to the woman to blame her for faults existing in the 
husband. The writer does not wish to be interpreted as stating that sterility 
does not frequently exist in women, but he does desire to emphasize the point 
that many more men are sterile than is generally supposed and that the sterile 
women are in this condition largely through the results of gonorrheal infection 
through their husbands. 1 

- Spermatozoa may be absent from the semen during convalescence from 
acute febrile conditions, valvular heart disease, and in general conditions associ- 
ated with lowered nutrition. On the other hand, the constant presence of 
spermatozoa in the urine, as well as in the semen, may be noted as a result of 
various pathological conditions as well as of venereal excesses or masturbation. 
To this condition is given the name of spermatorrhea. 

Medicolegal Aspects. 

Not infrequently the physician is called upon to decide whether certain 
stains are due to spermatic fluid or whether assault has been committed. If 

1 See Reynolds, Jour. Am. Med. Assn., 1913, LXI, 1363; Ibid., 1915, LXV, 1151; Dozzi, 
Gazz. d. osp., 1914, XXXV, 2069. 



SECRETIONS OF THE GENITAL ORGANS. 379 

the question is one of suspected rape, an examination of a drop of the vaginal 
fluid or of scrapings from the vulva or vagina will usually reveal the spermatozoa. 
Of course other signs, which are important from the medicolegal point of view, 
will be observed in the examination of the external organs. 

The stains usually subjected to medicolegal examinations for the presence 
of spermatic fluid have a grayish-yellow color, their size is somewhat variable, 
their contour usually irregular, and the linen upon which the stain is usually 
found is almost as stiff as if it had been starched. As spermatozoa are very 
resistant to the action of reagents as well as to putrefactive processes, they 
may be detected many years after the stain was made. It is, therefore, almost 
an impossibility to say by examination of a stain anything about the length of 
time the stain has been upon the cloth. 

A fragment of the linen, which shows the stain, is placed in a watch- 
glass and allowed to soak for one hour in 30 per cent, alcohol or in faintly 
alkaline water. It is then lifted from the solvent, placed in another watch- 
glass, and teased with needles in a solution of 1 per cent, eosin in glycerin. 
A few drops of this mixture are then placed upon a glass slide, covered with 
a cover-glass, and examined with a high-power dry lens. Spermatozoa, if 
present, will practically always be seen by this method. The heads are stained 
a deep red while the tails, which are usually broken off by the teasing, show 
a light reddish tint, which distinguishes them from the unstained vegetable 
fibers. 

Florence's Test. 

It not infrequently happens that spermatozoa may not be found, although 
the stain be due to spermatic fluid. The principle of this test is that spermatic 
fluid when treated with a solution of iodin in potassium iodid gives crystals 
which were supposed to be iodospermin. According to Bocarius, this substance 
is not iodospermin, but an iodin compound of cholin. This test would be 
given, therefore, by any substance containing cholin and cannot, for this reason, 
be distinctive for spermatic fluid. Such being the case, a negative result is 
of far greater importance than is a positive one. 

Technic. 

The reagent used consists of 1.65 grams of iodin and 2.54 grams of 

potassium iodid dissolved in 30 c.c. of distilled water. If a drop of spermatic 

fluid or of an aqueous extract of a suspected stain be treated with a drop of 

this solution and rmmediately examined under the low-power lens, long rhombic 

platelets of a dark brown color, fine needles, lance-shaped or ovoid bodies 1 

often grouped in rosettes may be observed. A positive reaction is seen many 

years after the formation of the stain so that a positive result is of value when 

other sources of cholin are excluded. 
t 
Barberio's Test. 

Barberio has found that the treatment of a drop of spermatic fluid or an 

aqueous extract of a suspected stain with a saturated aqueous solution of 

1 See Lecha-Marzo and Conejero, Semana Med., 1913, XX, 74; also Maestre and Lecha- 
Marzo, Ibid., 1914, XXI, 776. 



380 DIAGNOSTIC METHODS. 

picric acid gives immediately a precipitate of sharply refractile, yellow, ovoid 
or needle-shaped crystals which gradually increase in size. This test was 
supposed to be of much greater diagnostic importance than that of Florence, 
but the recent work of Fraenkel and Miiller has shown that the crystals are 
not sufficiently characteristic to permit of an absolute diagnosis. They call 
attention to the fact that substances other than spermatic and prostatic fluids 
may give similar crystals, but that in such cases these crystals are isolated 
and form usually on the border of the drop, while with spermatic fluids the 
crystals are numerous and are formed throughout the specimen. These 
workers recommend this test for the recognition of prostatic secretions or 
for the condition of azoospermatism, but caution the worker against making 
an absolute diagnosis from its presence in medicolegal cases. A negative 
result does not necessarily exclude the presence of semen. 1 

It will be seen, therefore, that neither one of the microchemical tests 
given above should be regarded as absolutely indicative of the presence of 
semen. It is much better practice to make repeated search for spermatozoa 
than absolutely to identify a stain as semen by the microchemical method. 

II. Female Secretions. 

(1). Vaginal Secretions. 

The normal secretion of the vagina is scanty, usually just sufficient to 
moisten the mucous membrane. It is clear or occasionally opalescent, semi- 
liquid in character, and is composed largely of mucus and epithelial masses. 
Its reaction appears variable. As a rule, it should be considered acid in the 
case of virgins, while in those who have borne children it is usually alkaline. 

Little is known regarding the chemical properties of this secretion. From 
the clinical standpoint the normal vaginal secretion is of importance owing 
to the fact that it possesses marked bactericidal properties. According 
to Kronig, pus organisms introduced into the vagina of pregnant women 
disappear in from four to thirty-six hours. Whether this bactericidal power 
is due to the reaction of the secretion or to some unknown agent is unsettled. 
A remarkable fact seems to be that frequent irrigation of the vagina with water 
or antiseptic solutions decreases the bactericidal power. If this be true, it 
is questionable whether frequent douching is advisable. 2 

Microscopic Examination. 

Outside of the great number of large, irregular, stratified, squamous 
epithelial cells which are constantly found in the vaginal secretion, one observes 
mucous corpuscles, a few large mononuclear leucocytes, cellular debris, and 
numerous bacteria. The bacterial flora 3 of the vagina is very extensive. 
These organisms are normally saprophytic and rarely take on pathologic 
functions, but they may occasionally do so. Among these bacteria we find 
the colon bacilli, streptococci, staphylococci, and bacilli which are not unlike 

1 See Lecha-Marzo, Arch, internat. de Med. leg., 1913, IV, 341. 

2 See Zweifel, Monatsschr. f. Geburtsh. u. Gynak., 1914, XXXIX, 459. 

3 See Kiister, Kolle and Wasserraann's Handbuch, 19 13, VI, 458. 



SECRETIONS OF THE GENITAL ORGANS. 381 

true diphtheria bacilli. None of these organisms are particularly important 
from the clinical standpoint and will be passed with mere mention. We 
do find, however, certain organisms which give rise to no particular clinical 
symptoms, but which are extremely confusing in the examination for the presence 
of the gonococcus. As will be learned from the later discussion of this organ- 
ism of Neisser, it appears in the form of biscuit or coffee-berry shaped 
diplococci, which are both intra- and extracellular and do not stain by 
Gram's method. In the early cases the intracellular organisms are few in num- 
ber. Saprophytic cocci often have the same morphology as the gonococcus, 
but are slightly larger, are variable to Gram's stain, and are rarely intracellular. 
They are, however, differentiated by the fact that they grow easily upon ordi- 
nary media, while the gonococcus requires special media. The more or 
less normal presence of these saprophytes should be constantly in mind and 
a diagnosis of gonorrhea made only when clinical symptoms are present to 
point to the gonococcus. It is wise in all doubtful cases to resort to cultivation, 
as one may very much regret having made a diagnosis of gonorrhea when such 
did not really exist. The practitioner should be cautioned to take his smear 
high up, in the vagina, preferably from the region of the cervix uteri. If this 
be done as a routine fewer specimens will be found showing these confusing 
saprophytes. Smears are frequently sent to laboratories for examination which 
will not show the gonococcus even though present in large numbers in the vagina. 
It is not sufficient to take a specimen from simple purulent material which 
maybe present in the lower portion of the vagina as the organisms are frequently 
absent in these locations. 

Pathology. 
Blennorrhea. 

Physiologically, an increased vaginal secretion (blennorrhea) is seen during 
sexual excitement, preceding menstruation, and during pregnancy, when a very 
profuse secretion may be observed. If this secretion contains a large number 
of epithelial cells and leucocytes, as seen in nonspecific inflammations, it 
becomes more or less creamy in color and is then called leucorrhea. 1 This 
type of leucorrhea should be sharply differentiated from the true pus secretions 
observed in the blennorrhagia of gonorrhea, as the former is not necessarily 
associated with pus formation. In pregnancy a slight catarrhal vaginitis is 
not infrequent, so that leucorrhea is more apt to appear at such times. If 
the inflammatory process becomes intense, large shreds of epithelium may 
be found and ulceration followed by vaginovesical or vaginorectal fistulae 
may be observed. Such pathologic findings are usually the result of gonorrhea. 
In slight catarrhal conditions of the vagina yellowish-gray patches may be 
seen, which are due to infection with mycotic fungi. 

It is not infrequent to find the trichomonas vaginalis in the vaginal prepara- 
tions. This organism has been previously discussed in the section on Feces, 
to which the reader is referred. The oxyuris vermicularis as well as its ova 

1 See Curtis, Surg., Gyn. and Obs., 1914, XVIII, 299; Ibid., 1914, XIX, 25. 



382 DIAGNOSTIC METHODS. 

have been reported in the vaginal discharge, but do not seem to have excited 
any pathologic changes. 

Purulent Secretions. 

True purulent secretions arising from the vagina are almost always due 
to the presence of the gonococcus. 1 This organism is accountable for a large 
number of gynecological conditions, so that it is wise for the practitioner to be 
able to identify it both from its clinical manifestations as well as by its laboratory 
detection. 

In doubtful cases cultures should be made and a portion of the pus dropped 
into the eye of a rabbit. The gonococcus itself may later be recovered from 
the conjunctival exudate. 

It is not to be assumed that the finding of the gonococcus in the vaginal 
discharge is necessarily evidence of a gonorrheal vaginitis or vulvovaginitis. 
It has been shown that the semen of the male as well as the urethral discharge 
may contain gonococci and that these may be introduced into a perfectly 
normal vagina without necessarily setting up gonorrhea. However, this is 
unusual. The gonococcus may arise from the urethra, the Bartholin glands, 
acute inflammatory processes of the uterus, or from a ruptured pyosalpinx. 
In any of these cases the gonococcus may be found in the vaginal discharge, 
so that the laboratory worker should be guarded in his diagnosis of a vaginitis. 
Further, suppurative processes which were originally due to the gonococcus 
may later take on a type of mixed infection or even become of the sterile type. 
This latter condition is especially observed in old Bartholinitis, metritis, and 
cystic salpingitis. The organisms usually associated with the gonococcus 
in the mixed infection are the streptococcus, staphylococcus, colon bacillus 
and pseudodiphtheria bacillus. In chronic metritis or salpingitis it is not 
infrequent to find the tubercle bacillus as the causative agent. 

Fetid Secretions. 

In these conditions the pus ic usually chocolate colored, has a fatty ap- 
pearance, is extremely repulsive, is frequently sanguinolent and contains 
numerous degenerated cells as a result of marked leucolysis. This condition 
is especially observed in puerperal infection and may be extremely severe. 

(2). Uterine Secretions. 

Normally, the uterus has no secretion beyond a slight mucoid one which 
is recognizable clinically. In inflammatory conditions, during normal men- 
struation, or following abortion or parturition, certain types of discharges 
are observed which have some clinical importance. 

Menstruation. 

Under normal conditions the menstrual fluid is at first mucoid in char- 
acter, but within a short time red cells appear and later the discharge takes 
on almost the character of pure blood. This menstrual fluid should be bright 
red in color, should contain no clots and should be discharged without causing 
active pain. This fluid contains red cells, leucocytes, and prismatic epithelial 

1 See Sharp, Jour. Infect. Dis., 19 14, XV, 283. 



SECRETIONS OF THE GENITAL ORGANS. $&3 

cells showing large areas of fatty degeneration. The duration of the men- 
strual period is variable, running between two and five days. The amount 
of blood lost under normal conditions averages about 200 grams, but may be 
much larger under pathologic conditions. Not infrequently do we find cases 
in which menstruation is associated with marked pain during more or less 
of the period of flow. This condition is known as dysmenorrhea and may be 
associated with the exfoliation of large shreds of mucous membrane, in some 
cases reported these shreds constituting almost a cast of the uterine cavity. 
To this latter condition is given the name membranous dysmenorrhea. For 
the pathologic significance of these abnormal types of menstruation as well 
as for a discussion of the condition associated with failure of menstruation 
(amenorrhea) the writer must refer to works on gynecology. 

The Lochia. 

By this term we have reference to the discharges from the uterine cavity 
during the puerperium. At first such discharges consist of blood, which 
may be in the form of clots, and decidual shreds along with epithelial cells 
which are probably of vaginal origin. This type is known as the lochia rubra 
or omenta. During the next two or three days the discharges become paler 
and thinner, the red cells diminish and the leucocytes increase, while the 
decidual shreds may continue approximately the same. This type is known 
as the lochia serosa. After about a week the discharge assumes a grayish or 
yellowish color and a creamy consistency, the red cells diminishing rapidly 
and the white cells increasing markedly. Microscopic examination shows, 
besides the leucocytes and epithelial cells, numerous fat globules and cholesterin 
crystals. This discharge may continue during the remainder of the period 
of uterine involution and is known as the lochia alba. Under normal condi- 
tions the lochial discharge has a faint odor, but is never fetid. If a portion 
of the placenta or membranes have been retained, the lochia may assume 
a dirty brownish color and become extremely fetid. After the first two or three 
days numerous bacteria, such as staphylococci, streptococci and colon bacilli, 
may be present, but no untoward symptoms exist unless these, along with other 
saprophytes, give rise to a distinct puerperal infection or sapremia. 

Amniotic Fluid. 

This is a thin, whitish or pale-yellow fluid containing the constituents 
of ordinary transudates. The reaction is neutral or faintly alkaline, the 
specific gravity varies between 1002 and 1008, and the amount of solids rarely 
reaches 2 per cent. The albuminous bodies are principally vitellin, serum 
albumin, and traces of mucin, while glucuse is absent. Urea and allantoin 
are present in traces, while creatinin has occasionally been reported. 

The amount of amniotic fluid varies between 700 and 1,000 c.c. Under 
pathologic conditions, however, this amount may be increased or decreased, 
giving rise on the one hand to polyhydramnios or dropsy of the amnion and 
on the other to oligohydramnios. For a discussion of these pathologic varia- 
tions as well as for a treatment of the subject of pathologic changes in the 
membranes, the writer will refer to works on obstetrics. 



3^4 



DIAGNOSTIC METHODS. 



Abortion. 

The recognition of abortion is usually made by examination of the material 
discharged from the uterine cavity. Usually one finds blood-clots in which 
the villi of the chorion are present. These usually appear as club-shaped 
masses with epithelial coverings, showing the characteristic capillary network. 
Moreover, decidual cells are usually present, and may be recognized by their 
large size, their round, polygonal, or spindle-shaped form, and their irregular 
and large nuclei with nucleoli. 1 




Fig. 116. — Chorionic villi. (McMurrich.) 

Vesicular Mole. 

This condition has been called dropsy of the villi of the chorion, hydatidi- 
form degeneration of the chorionic villi, cystic mole, and myxoma of the placenta. 
One of its most important symptoms is the expulsion through the vagina of 
the vesicles forming the degenerated mass. The mole is a mass of peduncu- 
lated vesicles which closely resemble a bunch of grapes or gooseberries. Each 
vesicle may vary in size from a millet seed to a large hazelnut and contains 
a fluid which is usually colorless and limpid, but may be reddish and some- 
what dense. Microscopic examination of the tissue shows the peculiar myxo- 
matous degeneration of the chorionic villi. 2 

Carcinoma. 

It is not infrequent to observe, in cases of severe hemorrhage through 
the vagina, the appearance of occasional shreds, which on microscopic exami- 
nation show the characteristic appearances of carcinoma of the cervix or body 
of the uterus. The diagnosis of carcinoma, however, would better be made 
upon sections removed by the surgeon rather than upon shreds found in the 
hemorrhagic fluids. Although a diagnosis may be at times possible, it should 
be somewhat guarded unless the clinical symptoms are distinctive. 

1 See Lackner, Surg., Gyn. and Obs., 1915, XX, 537. 

2 See Westermark, Hygiea, 19 14, LXXVI, 801. 



CHAPTER VIII. 
THE BLOOD. 

I. General Considerations. 

The blood is perhaps the most important tissue in the body, inasmuch 
as it is at once the purifier and the nutritive source of the cell. Any normal 
or abnormal product of cellular activity finds its way ultimately into the 
blood, either to be taken up by the assimilatory organs or to be thrown out 
by the excretory ones. While but relatively few disease processes are as- 
sociated with diagnostic findings in this tissue, yet many are characterized 
by definite manifestations which are invaluable aids to the clinician. 

It is, therefore, of the utmost importance that we should have a proper 
knowledge of the normal blood in order better to understand the various 
phases which characterize abnormal blood and which give to hematology 
such an interesting and important position in diagnosis. While it is true 
that some of the methods involved in hematological examinations require 
definite apparatus and a large experience for their proper interpretation, yet 
the results obtainable from the ordinary routine blood examinations are so 
invaluable, being in some cases pathognomonic, that no practitioner should 
consider himself fitted to give his patients the proper service without being 
equipped with a clear working knowledge of the methods of examination and 
the findings of normal and of abnormal blood. 

It is essential to remember that certain physiologic as well as pathologic 
conditions influence the quantity and quality of the blood. So great are the 
effects of digestion, exercise, nervous factors, massage, cold, heat, sweats, 
dysentery, constitutional and specific diseases, that one does not wonder at 
the many conflicting reports of cases showing widely varying hematological 
findings. As Grawitz has pointed out, no conclusion should be drawn from 
an examination of the blood without taking into consideration the physiologic 
and pathologic condition of the patient. 

It is a pleasure to observe in these days the tendency toward a more 
rational and thorough study of the plasma, the so-called " intracellular fluid" 
of the blood. We have forgotten, in our enthusiasm over the many valuable 
findings obtained from histological investigations, that the relations of the fluid 
portions of the blood are, in some cases, of quite as much importance as are 
the variations in the cellular elements. It is necessary only to cite the work 
on lysins, precipitins, agglutinins, opsonins, etc., to show the value of a more 
extended study of the plasma or serum. 

Regarding the technic of blood examinations, the writer will have much 
to say later, but he wishes to impress upon his readers one point which has 

25 385 



386 DIAGNOSTIC METHODS. 

been well expressed by Turk, namely, an indispensable basis for the proper 
utilization of any diagnostic, prognostic, or therapeutic method of clinical 
examination is a knowledge of the absolute limitations of the method. Reliable 
results can, however, be obtained only by those who are thoroughly familiar 
with the principles as well as with the technic and the little " knacks" of 
the method used. It will be found, when the attempts are made to apply the 
methods outlined, that quite as much depends on the exactitude with which 
the separate details are carried out as upon the selection of the method itself. 
It is not to be expected that a first trial will yield exact results or that a few 
determinations will perfect one in the methods of examination. Experience 
is the only teacher that can equip one with the skill and power of interpretation 
necessary to cope with the many difficulties to be overcome in the hematological 
investigations. 

II. Physiology and Chemistry. 

(i). Blood Formation and Blood-forming Organs. 

While it is impossible in a general work of this character to go into 
great detail regarding the formation of the blood, yet it seems to the writer 
that a brief discussion of this subject is extremely valuable both to the student 
and practitioner. 1 

Red Corpuscles. 

According to Kolliker, the first blood-corpuscles have their origin, in 
embryonal life, in the embryonic heart and blood-vessels. They appear 
as nucleated colorless cells, which later develop into colored corpuscles by 
the appearance of hemoglobin in some of the cells of the mesodermal 
cord, which cells go to form the first capillaries. Upon the formation of 
these vessels the cells lie within them as nucleated reds. At this time there 
are no true leucocytes and none appear until after the complete formation 
of the red cells, which is advanced as an argument against Pappenheim's 
theory of single origin of red and white cells. It will thus be noted that the 
vessel wall and the primitive erythrocyte have a common origin in the meso- 
dermal cord, the peripheral cells going to form the endothelium of the vessel 
and the internal cells the corpuscles. Up to the end of the fourth or fifth 
week of embryonal life all of the red cells are nucleated; while from that time 
on the relation of the nonnucleated to the nucleated forms gradually increases 
until at birth few if any nucleated cells obtain. 

In later embryonal life (about the third month), the liver becomes the 
chief seat of blood formation. During the fifth month, the spleen 2 and lymph- 
glands take up this work, and finally the bone-marrow becomes the seat 
of such activity. 

In extrauterine life, the bone-marrow is the chief point of formation of 
the red cells, but under pathological conditions the spleen and liver may 
assume their embryonic functions. It appears that the formation of nucleated 

x See Gruner, The Biology of the Blood Cells, New York, 19 14. 

2 See Morris, Jour Exper. Med., 1914, XX, 379; Hirschfeld and Weinert, Berl. klin. Wchn- 
schr., 1914, LI, 1026. 



THE BLOOD. 387 

reds in the adult is practically the same as in the embryo and that, at all periods 
of life, the red cell is the product of several series of mitoses of a colorless 
mesoblastic cell. The difficulty of tracing this series, from the large nucleated 
red cell to the colorless mesoblastic "mother-cell" in the marrow, has given 
rise to the diverse opinions now held regarding the ultimate development 
of the red corpuscles. 

Leucocytes. 

The earliest indications of the formation of leucocytes are seen in the 
presence of primary wandering cells, of mesodermal origin, which are found 
principally in the loose connective tissues of the early embryo. Though of 
mesodermal origin they are, from the first, quite distinct in morphology 
and, apparently, in function from the capillary endothelium and fixed con- 
nective-tissue cells. Their development has been traced by Ziegler to masses 
of mesodermal cells surrounding the cords from which the capillaries are 
formed. It thus seems that originally the parent leucocytes lie outside the 
vessels, into which they make their way by virtue of ameboid powers. 

Most observers find that the primary wandering cells produce, by mitotic 
division, one or more generations of colorless cells which gradually approach, 
in morphology, the early basophilic leucocytes of the circulation. Denys, 
Lowit, Ziegler, von der Stricht, and others claim that red cells and white 
cells develop from separate series of cells, which have become differentiated 
from the primary mesodermal cells with the first appearance of blood and 
blood-vessels. Kostianecki, Miiller, Schmidt, Saxer, Pappenheim, and others 
believe that the primary wandering cell persists in the blood-forming organs 
as the parent of both red and white cells. 

Before the leucocytes begin to appear in the circulation, mitotic figures 
are abundantly seen in the primary wandering cells in various situations. 
These are gathered in groups, first in the loose connective tissues of various 
regions, where lymph nodes subsequently develop; but the chief seat of the 
production of the leucocytes is found in the embryonal liver. In both situa- 
tions the wandering cells are found in the lymph and blood capillaries, in the 
interstices of the connective tissues, and between the liver cells. In later 
embryonal life the process is gradually transferred from the liver to the lym- 
phoid and adenoid tissues, as indicated by the development of lymph nodes, 
spleen, marrow, and thymus. Under normal conditions, the reproduction 
of leucocytes, in the adult, is limited to the lymphoid structures both of the 
lymph-glands and bone-marrow. 

(2). Total Volume of Blood. 

The various methods which have been advanced for the estimation of 
the total quantity of blood in the body are subject to such wide variations 
that they have yielded little exact information regarding this subject. The 
procedures advocated by Valentine, Vierordt, Buntzen, and Thibault have 
an error sufficiently great to exceed the physiological and pathological varia- 
tions of the blood. By these methods, the quantity of blood in the body 
has been estimated as equal to one-thirteenth of the body-weight. The so- 



388 DIAGNOSTIC METHODS. 

called clinical methods of Quincke or of Tarchanoff are of purely theoretical 
interest, because certain factors, such as the appearance of the patient or the 
volume of the pulse, are taken into consideration in making a rather unreliable 
guess as to the total quantity of blood in the body (Buckmaster). 

By the use of a reliable method, introduced by Haldane and Smith, 1 the 
total volume of blood may be fairly accurately estimated. This method is 
based on the following points. The capacity of hemoglobin for oxygen and 
for carbon monoxid is identical. On the assumption that none of this latter 
gas is oxidized in the body, and that no substance in the blood, other than 
hemoglobin, unites with it, the experimenter is able to determine the CO 
capacity and hence the O capacity of the blood. This method has, however, 
little clinical application and will be left with reference to the original work. 
In the cases studied by Haldane and Smith by this method, the average 
value was 3,240 ex. or, on the basis of a specific gravity of 1,060, about 3,434 
grams. This yields, according to Smith, a figure ranging between one-six- 
teenth and one-thirtieth of the body-weight. 2 In obese persons the volume 
of blood is less, proportionately, than in the more normal specimens of 
mankind. 

The question of the volume of blood in the body is of great importance 
in the study of the changes taking place in this tissue. It must be remembered 
that the number of red or of white cells in a cmm. of blood will depend upon 
the total amount of blood present. If for any reason the volume is diminished 
or increased, corresponding changes, in the inverse sense, will be observed 
in the number of the cellular elements per cmm. It seems to the writer, there- 
fore, that certain factors, not ordinarily taken into account in blood examina- 
tions, should be known before any definite report is made upon a blood count. 
It may be readily seen that a concentration of the blood, due to hemorrhage, 
diarrhea, sweating, etc., will lead to an apparent increase in the number of 
corpuscles. 

Certain physiologic and pathologic conditions lead to definite changes 
in the volume of the blood, as such, or of some of its constituents. As Plehn 
has recently shown, the volume remains quite constant or is adjusted through 
the activity of the capillary endothelium and through the influence of the 
nervous system. However, definite changes of a more or less transitory nature 
do occur and exert marked influences on the results of blood examinations 
as well as upon many pathologic conditions. 3 

Oligemia. 

By this term is meant a reduction in the total volume of blood, both as 
regards the liquid and the cellular portions. This condition is most frequently 
noticed after profuse hemorrhage and may be so marked that death results. 
In other cases, in which the hemorrhage is less extensive, the loss of blood is 

1 Jour. Physiol., 1896, XX, 497; Ibid., 1900, XXV, 333. 

2 Keith, Rowntree and Geraghty (Arch. Int. Med., 1915, XVI, 547) consider that the blood 
constitutes 8.8 per cent. (1/11.4) of the body-weight. Normal individuals have approxi- 
mately 85 c.c. of blood per kilogram. 

3 See Kammerer and Waldmann, Deutsch. Arch. f. klin. Med., 1913, CIX, 524; Miller 
Keith and Rowntree, Jour. Am. Med. Assn., 1915, LXV, 779. 



THE BLOOD. 389 

made up by osmosis from the lymph spaces into the capillaries and, later, 
by an increase of the cellular elements due to compensatory activity of the 
hematopoietic organs. 

Plethora. 

The opposite of the preceding condition is known as plethora, a state 
characterized by an increase in the total volume of blood. There has been 
much discussion as to whether a true plethora exists, but there can be little doubt 
that a transitory plethora vera may occur as a result of direct transfusion of 
blood, and also, according to Bergmann and Heissler, who have established 
the fact that there is a direct ratio between the volume of blood and the size 
of the heart, on the one hand, and the muscular development of the subject, on 
the other, as the result of increased muscular activity, provided the loss of 
fluid by perspiration is not excessive. Such a plethora disappears, of course, 
in a very short time. 

In this discussion we must distinguish between a serous and a cellular 
plethora. By the former is meant an increase in the volume of blood due 
to excessive quantities of its liquid and soluble constituents; while by the latter 
we understand an increase in the number of corpuscular elements, that is a 
polycythemia. Serous plethora is most frequently observed in organic 
lesions of the kidneys and of the heart, in which a diminished elimination of 
water and inorganic constituents is noted. This condition is usually of a 
transient duration, as the volume of blood is soon restored to normal by intra- 
capillary transudation and by diffusion. Osmotic effects must also be taken 
into consideration here, inasmuch as the salts will tend to diffuse out from the 
blood and will consequently draw water after them, giving rise, under certain 
conditions, to dropsical effusions of a more or less transient duration. 

Hydremia. 

Another condition of the blood is frequently observed, in which an in- 
crease in the quantity of the liquid constituents is observed. This is known 
as hydremia and is different from serous plethora, as the latter carries with 
it an increase in the saline as well as in the watery portion of the blood. In 
hydremia the specific gravity of the blood is reduced, while in serous plethora 
it is increased. As Engel 1 has shown, the estimation of the coefficient of 
refraction of the blood serum is a reliable method for the clinical study of the 
subject of the water content of the blood. 

Hydremia may be produced by any factor which changes the normal 
relationship of the blood constituents in such a way that the watery portion 
is relatively increased. It is in these more or less physiologic states that we 
are apt to observe the greatest variation in the blood counts. As the cellular 
elements are not simultaneously increased, the drop of blood under examination 
contains relatively fewer cells than normally. The most common physiologic 
causes of hydremia are the ingestion of large quantities of fluids, saline trans- 
fusions, and vasomotor dilatations as a result of exercise or nervous influences. 

1 Magyar Orvosi Arch., 1906, VII, 555. 



39° 



DIAGNOSTIC METHODS. 



In severe anemias we find the watery portion of the blood relatively increased. 
In dropsical states, following cardiac or renal lesions, we often observe such a 
condition, whose duration will depend, of course, upon the etiological factors 
of the trouble. 

Anhydremia. 

This is a condition characterized by a diminution in the liquid constituents 
of the blood. There is no change in the cellular elements and hence a blood 
count will show an erroneous increase in the number of cells. In this condi- 
tion the specific gravity of the blood is naturally increased. 

Anhydremia follows any condition which results in the excessive loss 
of fluid from the body, as, for instance, that following profuse diarrhea, polyuria, 
sweating, vomiting, and effusions into the various serous cavities of the body. 
According to Oliver, this state may be caused by influences which increase the 
arterial tension and hence bring about an increase in the passage of water from 
the vessels into the tissues. Thus, for instance, we may observe anhydremia 
following local and general exercise, massage, bathing, etc. 

(3). Volume Relations of Cells to Plasma. 

The study of the relationship between the cellular and the intracellular 
portions of the blood is a comparatively recent addition to the technic 
of blood examinations. This determination is based on the principle that the 
corpuscles may be thrown by centrifugal force to the distal end of a calibrated 
tube, while the plasma will collect in the proximal portion. If the tube be 
properly calibrated, the percentage relations of the cells and plasma may be 
readily ascertained. These ideas were used by Hedin in elaborating the earlier 
methods of Blix. 






m 



Fig. 117. — Daland's hematocrit. 

Daland's Hematocrit. 

D aland has introduced a modification of the clumsy model of Hedin and 
has succeeded in simplifying the technic to such an extent that this method 
is directly applicable to clinical use. His instrument is shown in the accom- 
panying cut. 

One of the calibrated glass tubes is fitted with the rubber tubing and is 
filled with blood from the ear or finger. The forefinger, smeared with a 
little vaselin, is then placed over the beveled end of the tube and the rubber 
tubing withdrawn. Insert the tube into one arm of the frame, the other arm 
of which should carry the second tube filled in the same manner, in order to 
balance the instrument and to control the reading. Rotate the spindle for 
three minutes at such a rate of speed as will insure 10,000 revolutions per 



THE BLOOD. 391 

minute (80 revolutions of handle). In this way the corpuscles are separated 
from the plasma and are distinguishable as a distinct column, which may be 
read off directly from the graduations of the tube. These divisions will give 
the percentage relations of the cells and plasma as the tube is divided into 
100 equal portions, each division of the scale representing approximately 
100,000 cells. This latter makes it possible to make a rather rough blood 
count with this instrument, but it is to be remembered that accurate results 
cannot follow, as we find such variations both in the size and elasticity of the 
cells in the different conditions in which the number of cells is most sought. 




Fig. 118. — Hematocrit tube. 

Volume Index. 

Capps 1 has introduced the conception of volume index, that is the 
quotient of the volume per cent, as obtained with the hematocrit, and the 
blood count in terms of per cent. Sahli advises the use of the expression 
volume quotient or volume value for this factor. 

By means of the hematocrit the volume of the red cells, as compared with 
that of the whole blood, is taken. In normal cases this is about 50 per cent, 
which is reckoned as one. Hence the volume of the red cells may be obtained 
directly in percentage value. The red cells are then counted by the method 
to be later outlined, and the result is expressed in percentage by comparing 
this count with a so-called normal one of 5,000,000 red cells. By dividing 
the volume of red cells (in per cent.) by the per cent, of red cells, Capps obtains 
his volume index of the red cells. 

In normal conditions this quotient is one. According to Capps, an 
increase of this index is a constant factor in pernicious anemia. The color 
index never exceeds the volume index in such cases, which fact shows that 
there is no supersaturation of the corpuscles with hemoglobin. In primary 
and also in secondary anemia this factor is diminished. Here we find the 
color index often falling below the volume index. 

This method may be used in detecting various pathological conditions 
of the blood. According to Emerson, it is used in the Johns Hopkins Hospital 
in ascertaining the presence of lipemia, cholemia, or hemoglobinemia. It would 
seem to the writer that it could be employed with advantage as a routine 
procedure, especially in hospital practice. The osmotic pressure of the plasma 
plays a great role in this determination, as the concentration of the blood may 
be such as to cause swelling or shrinkage of the cells. As Capps has found, 
normal red cells with a volume index of one have their discoplasm saturated 
with hemoglobin. Hence, if the hemoglobin index becomes greater than one, 
an enlargement of the red cells is indicated. Upon the other hand, the color 
index may fall, regardless of a corresponding lowering of the volume index. 

1 Jour. Med. Research, 1903, X, 367. 



392 DIAGNOSTIC METHODS. 

It follows, therefore, that, if the color index is above normal, the volume index 
must also be increased; while, if the hemoglobin index is below normal, the 
volume index is not necessarily diminished (Sahli). 

(4). Methods of Obtaining Blood. 

The method of obtaining blood for examination depends upon the amount 
desired and on the examination which is to be made. For ordinary routine 
work only a few drops are necessary, while for bacteriological investigations 
2 to 20 c.c. may be required. 

In obtaining the blood no set rule can be laid down as to the proper place 
from which to take the specimen. We should select the part which promises 
the best results, avoiding naturally the points which are cyanosed, eczematous, 
edematous, hyperemic, cold, unduly heated, or, in other words, any part 
which is not normal. The ear usually furnishes the best results, in the writer's 
opinion, because its puncture is relatively painless, it is easily accessible (which 
point is often of importance in attempting to obtain blood from those who are 
comatose or who refuse to submit their hand for puncture), and because the 
patient, who may be easily affected by the sight of blood, can not see the drop. 
If the lobe of the ear is thick it is usually pricked on the flat side, but if it be 
thin it is well to make the puncture on the edge and parallel to the surface. 
Some workers prefer the palmar surface of the ball of the middle or second 
finger of the left hand and others advise pricking the arm over a small super- 
ficial vein. In cases in which repeated examinations of the blood are to be 
made the parts should be varied in order to prevent soreness and also to 
avoid anesthesias which occasionally follow repeated use of the same site 
of puncture. 

Quite a number of special forms of blood needles, as, for instance, those 
of Francke and of Daland, are on the market, and each of them has its ad- 
vocates. Many of them are in the form of special holders, which permit a 
puncture of a desired depth to be made without any danger of going too deep. 
These are to be recommended to those only who seem unable to control their 
stab. The writer does not find that the results obtained by the student are 
any better with such instruments than are those following the use of the ordinary 
Hagedorn surgical needle. In lieu of any better article, a clean steel pen with 
one nib broken will yield admirable results. The one thing to bear in mind 
in selecting a needle for blood work is that the point have a cutting- 
edge and should not be round or sharp nor should it be too long or slender. 

Having decided upon the part from which the blood is to be taken, this 
surface is washed carefully with water and alcohol or ether and is then thor- 
oughly dried. Avoid any undue rubbing as this will cause hyperemia and will 
introduce an error into the work. It is, as a rule, unnecessary to sterilize 
the needle but if it seems advisable to do so on account of the patient's 
attitude, the sterilization is best done by heat, hydrogen peroxid or alcohol, 
allowing the needle to cool before making the puncture. In this latter part 
of the technic, much depends on the amount of blood desired as regards the 
use of a short quick stab or a slow steady puncture. The latter procedure 



THE BLOOD. 



393 



iSiMMfijh 



usually yields more blood but is more painful. It is much better to prick 
the patient too deeply, going even through the lobe of the ear, than it is to 
subject him to repeated punctures. The part pricked should not be squeezed 
nor held in a position which will cause an abnormal circulation. If the 
puncture is successful, the blood will come out in good-sized drops. The 
first of these are wiped away and subsequent ones used for the examination. 
As often happens, the blood coagulates fairly quickly, so that the coagulum 
should be wiped off with a little alcohol followed by a dry cloth. It is im- 
portant to remember that a patient with hemophilic tendencies or history 
may bleed very easily from a very slight puncture. Care should, 
therefore, be taken to question the patient regarding the ease 
with which blood flows from a wound and also regarding the his- 
tory of " bleeders" in the family. 

If considerable blood is desired, resort must be made to venous 
puncture. The site of this operation is usually the median basilic 
vein at the bend of the elbow. This vein may be made more 
prominent by tying a tight bandage around the arm, but the 
bandage should be removed before the blood is withdrawn, except 
when serum reactions are to be studied. The site of puncture 
must be thoroughly cleansed, using the precautions observed before 
any surgical operation. Likewise, the needle and the aspirating 
instrument must be absolutely sterile before puncture is made. 
The instrument best adapted for this purpose is, in the writer's 
experience, the Liier syringe, which is made of glass with a 
tightly fitting glass plunger and adjustable platinum needle. 
From 2 to 20 c.c. of blood are withdrawn and immediately 
placed in work. The question of the bacteriological examination 
and of serum reactions will be discussed in a later section. 

(5). Physical Properties. 

The blood must be regarded as a fluid tissue, consisting of a 
transparent liquid, the plasma or liquor sanguinis, in which are 
suspended the corpuscular elements, erythrocytes, and leucocytes. 
Besides these latter cells we find two constituents, the blood plates 
of Bizzozero and the hemoconien (blood dust), which are hardly fig. 119 — 
to be considered as true corpuscular entities. Blood needle. 

As it flows from the vessels, blood is a thick, viscid, red liquid, having 
a peculiar odor, a salty taste, and an alkaline reaction to litmus. If allowed 
to stand it shows, unless certain precautions are observed, the peculiar phe- 
nomenon of coagulation. In this process the blood is separated into two por- 
tions, the cellular elements and the plasma, the latter changing, as the process 
goes on, into serum and the clot (fibrin), which holds the corpuscles in its 
meshes. In the diagram given below, adapted by Webster and Koch, may 
be seen the composition of the blood. 



394 



Plasma 



Blood 




Serum 




Fibrinogen 



DIAGNOSTIC METHODS. 
Serum Albumin. 
Serum Globulin. 

Glucose, extractives, calcium salts, sodium 
and potassium chlorids, carbonates, phos- 
phates, etc. 
(yields fibrin). 



Cellular Elements 





Oxy-hemoglobin. 

Lecithin. 

Salts. 

•Fibrin Ferment. 



Red Corpuscles 

White Corpuscles' 
Blood Plates. 

Hemoconien. 

(A). Color. 

The color of the blood is due to the presence in the erythrocytes of an 
iron-containing albuminous substance, hemoglobin, which has remarkable 
affinity for oxygen and other gases. This latter property enables this pigment 
to play one of the most important roles in the body economy. Arterial blood 
is bright red in color, while venous blood shows a purplish-blue tint. These 
variations are due entirely to the relative proportions of oxygen and of carbon 
dioxid in the two types of blood. Many different shadings are observed, 
physiologically and pathologically, in the coloration of the blood, and each is 
due to some combination of hemoglobin with normal or abnormal substances. 

The presence of large numbers of red corpuscles in the blood gives rise 
to a characteristic opacity of this tissue. If, for any reason, such as admixture 
of blood with water, dilute salt solutions, urea, ether, snake venom, extract 
of mushrooms, etc., the blood loses its opacity, the change is due to the dis- 
solving out of the hemoglobin from the red cells. This is the well-known 
phenomenon of "laking" or, better, of hemolysis, about which we will have 
something to say later. 

The normal color of the blood is often changed in pathological conditions. 
Thus, in anemia the blood is pale and watery; in leukemia it may be milky; 
in diabetes buff-colored; while in poisoning with potassium chlorate it is choco- 
late-colored and in that with carbon monoxid it is bright red. 1 
(B). Odor. 

The odor of the blood is peculiar and indescribable. This halitus san- 
guinis is due to the presence of certain volatile fatty acids and may be rendered 

1 See Gaisbock, Med. Klinik, 1912, VIII, 1906. 



THE BLOOD. 395 

more distinct by the addition of concentrated sulphuric acid, which increases 
the volatility of these acids (Barruel's test). 

(C). Reaction. 

If we are to accept the teachings of physical chemistry, that the alka- 
linity of a solution is due to the presence of free hydroxyl (OH) ions and that 
its acidity depends on the surplus of free hydrogen (H) ions, we must grant 
that the blood is a practically neutral fluid. If, however, we have in mind 
the acid-combining power of the blood, we must regard the reaction of this 
tissue as alkaline. It is certain that the blood shows both acid and alkali 
combining powers due to the presence of protein constituents as well as to 
both acid- and alkali-reacting substances, the measure of such powers being 
dependent on the indicator used in the estimation. As the combining power 
for acids is greater, in the case of blood, than it is for alkalies, the reaction, as 
judged by titrimetric methods, must be alkaline. 

The normal free or diffusible alkalinity of the blood is due to the presence 
of disodium hydrogen phosphate (Na 2 HP0 4 ), sodium bicarbonate (NaHC0 3 ), 
and sodium carbonate (Na 2 C0 3 ). This total diffusible alkali constitutes, 
according to Brandenburg, about 20 per cent, of the entire alkalinity and may 
be measured by dialyzing against known alkaline solutions and observing the 
concentration at which the strength of the known solution does not change. 
This factor represents the so-called alkaline tension of the blood and remains 
fairly constant, in normal cases, at about 60 mg. of NaOH per 100 c.c. of blood, 
while in pathologic conditions, such as uremia, diabetes, etc., it is somewhat 
reduced. 

Besides this diffusible alkali, the blood contains nondiffusible alkali 
bound to the proteins. This portion represents normally about 80 per cent, 
of the total alkalinity, and is dependent largely on the cellular content of the 
blood, as the soluble protein constituents are not generally subject to wide 
variations. The fluctuations in this nondiffusible alkali are no doubt account- 
able for the great differences in the figures given for the alkalinity of the blood. 

The subject of the reaction of the blood is one which should furnish, if 
properly studied by reliable methods, much valuable data upon subjects 
which are now very obscure. Unfortunately, however, most of the methods 
at our command are so unreliable and so inexact that the results have little 
more than a comparative value. As Moore and Wilson 1 have pointed out, the 
titration methods do not give us the true neutrality of the blood, but rather 
the amount of alkali or of acid which may be added to it without raising the 
hydroxyl or hydrogen concentration above certain low limiting values. The 
reaction of the blood depends on the indicator used for the determination of 
the neutral point and cannot be definitely measured by any method which 
employs an indicator for such purposes. These writers have introduced the 
term "reactivity" to indicate the property, possessed by the blood, of com- 

1 Biochem. Jour., 1906, I, 297. See, however, Sellards, Bull. Johns Hopkins Hosp., 1914, 
XXV, 101. 



39^ 



DIAGNOSTIC METHODS. 




bining with both alkalies and acids in such a way as not to raise its ionic 
composition. 

In selecting the methods for the quantitative estimation of the so-called 
alkalinity of the blood, I limit myself to those of Dare 1 and of Salkowski, 2 
as neither depends upon the titration of the blood. The figures given by the 
older titration methods have such a wide range, being as low as 162 mg. and 
as high as 800 mg. of NaOH per 100 c.c. of blood, that the writer feels com- 
pelled to give them up for methods which seem to yield more exact and ac- 
ceptable results. 

Method of Dare. 

This method is based on the fact that the absorption 
bands of the spectrum of oxyhemoglobin disappear at the 
point of exact neutralization of the blood. Although it 
has not been conclusively proven that oxyhemoglobin is not 
destroyed before the point of complete neutralization is 
reached, yet the results with this method are rarely so 
variable as are those with the titration methods. 

The apparatus (hemoalkalimeter) may be seen in the 
accompanying illustration. It consists of a special gradu- 
ated tube (A), a blood pipet (B) which holds 20 cmm. 
(15 mg. by weight) of blood, a reagent pipet (D), and a 
hand spectroscope (E). 

The method of using this instrument is as follows: 
The tube (A), into which fits the pipet (B), is held hori- 
zontally in such a manner that the pipet may be filled 
by capillary attraction with blood from the finger or ear. 
With a medicine dropper, containing distilled water, the 
pipet is washed free of blood, using sufficient water to 
bring the diluted blood to the zero-point of the tube A. 
This diluted blood is then thoroughly mixed by shaking 
and inverting the tube, after which agitation it is allowed 
to settle. In this process care should be taken to close 
the opening (C) with the thumb to prevent loss of fluid. 
The reagent pipet (D) is then filled with the following test solution (N/200 
tartaric acid, each c.c. of which is equivalent to .0002 gram of NaOH) and is 
connected with the end of the blood pipet by means of rubber tubing. 

Tartaric acid, °-375 gram. 

Alcohol (95 per cent.), 100.000 c.c. 

Distilled water, q. s. ad., 1,000.000 c.c. 

By compression of the bulb of the pipet the reagent is forced through the 
blood pipet into the tube A. Mix the fluids by inversion, care being taken 
to avoid loss of fluid. Adjust the spectroscope to the tube below the zero 
point after each addition of reagent and observe the spectrum for the presence 

iPhila. Med. Jour., 1903, XI, 137. 

2 Centralbl. f. d. med. Wissensch., 1898, XXXVI, 913. 



81 



— A 



Fig. 120. — Dare's 
hemoalkalimeter: A, 
alkalimeter tube; B, 
automatic blood pi- 
pet; C, air hole; D, 
reagent pipet. 



THE BLOOD. 397 

of the two bands of oxyhemoglobin. This is best done by holding the tube a 
constant distance from an artificial light. This part of the technic is very 
tedious, but is necessary until the observer learns to associate the change in 
color of the mixture with the point of neutralization. If the spectroscopic 
bands of oxyhemoglobin still persist, more reagent is added and the mixture 
shaken as before, until these bands can no longer be seen. This is the point 
of neutralization and the end-point of the test. Read off, from the graduations 
on the tube, the number of c.c. of reagent used. 

The following table, representing the graduations on the tube, gives 
the equivalent of NaOH for every fraction of a c.c. of reagent used. It is 
calculated on the basis of 15 mg. of blood for every 2 c.c. of the N/200 tartaric 
acid. Dare considers the normal figure to be 2 c.c. of acid, which represents 
266 mg. of NaOH per 100 c.c. of blood. 



C.C. of 


Milligrams of NaOH 


reagent. 


per 


100 c.c. of blood. 


3.0 




376.0 


2.8 




360.0 


2.6 




345-o 


2.4 




319.0 


2.2 




292.0 


2.0 




266.0 


1.8 




239.0 


1.6 




212.0 


1.2 




176.0 


1.0 




*33-° 


0.8 




96.0 


0.6 




79.0 


0.4 




53-o 


0.2 




26.6 



The writer has found that this method is capable of easy application and, 
with a little practice, will give very reliable and constant clinical results. 
Naturally the personal equation arises in the reading of the spectroscope, but 
this is easily corrected by experience. As the spectroscopic changes are some- 
times slow in appearing, one should not be in too great haste to finish his 
determination. The figures obtained with this method are somewhat lower 
than those with the Salkowski technic and are probably more nearly repre- 
sentative of the nondiffusible than of the total alkalinity. 

Method of Salkowski. 

This method, like the preceding one, has the advantage of avoiding 
direct titration of the blood, but has the objection that a considerable time is 
necessary to obtain certain results. 

The method is very simple and is based on the principle that ammonium 
salts are decomposed, in the presence of alkalies, with the liberation of free 



398 DIAGNOSTIC METHODS. 

ammonia, which latter substance may be absorbed by a standard acid solution 
and determined by titration of the excess of acid. 

Twenty grams of finely powdered chemically pure ammonium sulphate 
(NH 4 ) 2 S0 4 are placed in the larger lower dish of Schlosing's apparatus (see 
Urine) and are dissolved in 20 c.c. of distilled water. Ten c.c. of N/ 10 sulphuric 
acid are placed in the upper dish. Pour into the lower dish, which contains the 
ammonium sulphate solution, 10 c.c. of blood, which has been measured in a 
cylinder previously washed with 1 per cent, solution of sodium oxalate to pre- 
vent coagulation of the blood. Mix the blood and ammonium sulphate solution 
thoroughly and cover the dishes as quickly as possible with a bell jar, which 
should be tightly fitted to the glass plate with vaseline. Allow the apparatus 
to stand at room temperature for five to six days, when the ammonia, liberated 
by the action of the alkali of the blood upon the ammonium sulphate, will 
have been taken up by the sulphuric acid. Titrate this acid solution with 
N/10 NaOH, using methyl orange or rosolic acid as an indicator, until the 
point of neutralization is reached. Subtract the number of c.c. of NaOH 
used in the titration from the original number of c.c. of H 2 S0 4 taken and mul- 
tiply the result by 0.004006, which will give the amount of sodium hydrate 
equivalent to the ammonia liberated from the 10 c.c. of blood. To obtain 
the amount of NaOH per 100 c.c, multiply the above result by 10. 

This method is appealed to on account of its simplicity and its reliability, 
being certainly more accurate than the methods of direct titration. According 
to Waldvogel, the normal values obtained by this method are 350 to 400 
mg. of NaOH per 100 c.c. of blood, being somewhat higher than those obtained 
with Dare's method owing to the fact that all of the alkali present, whether 
diffusible or nondiffusible, will react by this method. 

The reaction of the blood varies in certain physiologic states, as, for in- 
stance, during digestion. The alkalinity or the "reactivity to acids" is higher 
in man than in woman and the child; is at its minimum in the early morning, 
rises during the afternoon, and falls in the evening; is increased during digestion 
and falls after digestion is complete; is decreased by excessive exercise and also 
by a diet deficient in nitrogenous constituents. Many of the beneficial results 
of massage and baths are unquestionably due to an increase in this factor. 

It must be remembered that the normal balance between the acid and 
alkali constituents of the blood is easily maintained, otherwise the system 
would be in a state of constant disorder, subject as it is to the influence of acids, 
either produced within itself or taken into it from without. 1 As Moore and 
Wilson have shown, the susceptibility of the living cell to increase in concen- 
tration of either hydrogen or hydroxyl ions is due to the fact that the protein 
of the cells possesses strong affinity for both these ions, forming feebly disso- 
ciated salts with them. Increased alkalinity of the blood is supposed to 

1 See Lundsgaard, Biochem. Ztschr., 1912, XLI, 247; also, Marriott, Jour. Biol. Chem., 

1914, XVIII, 507; Hopkins, Lancet, 1914, I, 1589 and 1616; Lewis and BarcrofL Quart. Jour. 
Med., 1915, VIII, 97; Van Slyke, Stillman and Cullen, Proc. Soc. Exper. Biol, and Med., 1915, 
XII, 165 and 184; Hasselbalch and Gammeltoft, Biochem. Ztschr., 1915, LXVIII, 206; Levy, 
Rowntree and Marriott, Arch. Int. Med., 1915, XVI, 389; Menten and Crile, Am. Jour. Phvsiol., 

1915, XXXVIII, 225. 



THE BLOOD. 399 

denote increased antibactericidal power of this tissue. For this reason we must 
assume some direct relationship between immunity and alkalinity. Moreover, 
the further study of the opsonic, lytic, and other properties of the plasma may 
reveal definite influences of the alkalinity upon such processes. 

It has been found that pathologic changes are very often evident in the 
reaction of the blood. Thus, for instance, we find in severe secondary anemia 
and in pernicious anemia a marked decrease in the alkalinity, while in chlorosis 
we observe little or no change. The degree of alkalinity is reduced in prac- 
tically all infectious diseases, in uremia, in diabetes, and organic diseases of 
the liver, etc., while in chronic diseases it may, at times, show an increase. 

(D). Specific Gravity. 

The specific gravity of normal blood varies between, 1,055 an d ^oo^, 
the average being 1,060. Certain variations in this figure are observed depend- 
ing on the sex or age of the patient or upon the time and temperature at which 
the determinations are made. 

The most accurate method of determining the specific gravity is, of course, 
the use of the pycnometer. This method is open to the objection that it re- 
quires much more blood than can usually be obtained in routine work. In 
cases in which bleeding can be resorted to without detriment to the patient 
this method is the one to use, as it gives the most reliable and accurate results. 
The writer has used it in many cases of pneumonia, where the withdrawal 
of a certain amount of blood is often beneficial, and finds the results all that 
could be desired. Besides the quantity of blood (5 to 50 c.c.) which is required 
there is also necessary a very accurate chemical balance, else the results will be 
influenced by the inaccuracies in the weighings. 

The technic is as follows: weigh the pycnometer empty, filled with 
distilled water, and then filled with blood. Care should be taken to have the 
vessel absolutely dry and clean before weighing it empty and before filling 
with either water or blood. Subtract the weight of the empty bottle from 
that of the bottle filled with blood and divide this figure by the difference in 
weight between the bottle filled with water and the empty bottle. The result 
will be the specific gravity of the blood, water being taken as unity. We 
should be careful in this determination to have the temperature of the water 
the same as that of the blood in order to insure accurate results. 

Method of Schmaltz. 

This method is a modification of the above and does not give quite as 
accurate results. It consists in the use of small tubes, which hold about 
1/10 c.c. These tubes are constricted at the end to prevent loss of blood and 
are filled by capillary attraction. The determination is made in the same 
way as with the pycnometer. More or less manipulative skill is necessary 
in the handling of these tubes, but the results are sufficiently accurate for 
most purposes. 

The more frequently employed methods of determining the specific 
gravity are the so-called areometric ones. The principle of these procedures 
is the determination, by the use of accurate hydrometers, of the specific 



40o 



DIAGNOSTIC METHODS. 



gravity of a liquid mixture, in the center of which a drop of blood will re- 
main suspended. 

Method of Hammerschlag. 

This method is a strictly areometric one and consists in the use of a 
mixture of benzol and chloroform into which a drop of blood is introduced 
through a capillary tube. If the drop rises in the mixture benzol is added 
and if it sinks chloroform is used. The point at which the drop of blood 
remains suspended in the center of the perpendicular axis of the mixture is 
taken as the one representing the specific gravity of the blood. As the means 
of estimating the density of this mixture, we employ an accurately gradu- 
ated hydrometer. It must be remembered that the fluid mixture should be 
well stirred after the addition of either benzol or of chloroform in order to 
insure uniform density throughout. As the mixture evaporates rapidly we 
must work quickly and should confirm our results by a duplicate determina- 
tion. A further precaution should be to allow no bubbles 
of air to adhere to the drop. This is fairly well accom- 
plished by the use of the capillary blood pipet. It is also 
essential that the temperature of the mixture should not 
vary to any appreciable extent. This method is simple 
and, with the precautions mentioned, will yield good clin- 
ical results. 

The specific gravity of the serum may be tested in this 
same way, first allowing the blood to coagulate in sealed 
tubes and then drawing off a drop or two of the separated 
serum. 

Naturally, the specific gravity of the blood is a measure 
of its concentration and, hence, of its water content. We 
are, therefore, certain to find variations in this factor under 
the influence of any physiologic or pathologic changes, 
which are associated with fluctuations in the volume of blood. 
Physiologically, the specific gravity is higher in men than in women and 
children; is higher in venous than in arterial blood, and is lower after ingestion 
of large quantities of fluid in the food or after infusions. Clinically, we find 
that the specific gravity of the blood runs parallel to the number of corpuscles 
and to the amount of hemoglobin in the red cells. So striking is this ratio 
that it was formerly used to determine the percentage of hemoglobin in the 
blood. Any marked alteration in these constituents gives rise to a variation 
in the specific gravity. Thus, there is observed in anemia in which there 
is a lowered percentage of hemoglobin, and in those forms of secondary anemia 
which are characterized more particularly by diminution in the number of 
cells, a low figure for the specific gravity. In polycythemia, on the other hand, 
we find the specific gravity increased as a result of the increased corpuscular 
content of the blood. 

Pathologically, the specific gravity may run between 1,026 and 1,068. An 
increase is noted in practically all febrile diseases, in those conditions associated 




Pyc- 






THE BLOOD. 4OI 

with cyanosis, and in disorders leading to obstructive jaundice. In conditions 
showing marked diuresis, diarrhea, or sweating an increase is likewise observed, 
but such changes are usually of slight duration, as the blood soon adapts itself 
to the condition by withdrawing liquid from the tissues to compensate for the 
loss in the above processes. In nephritis we may find either an increase or 
a decrease in the specific gravity, depending on the osmotic changes which take 
place in this disease. 

(£). Viscosity of the Blood. 

Freshly drawn blood has a greasy feeling, which is replaced by a stickiness 
as coagulation proceeds. This viscosity or internal resistance of the blood 
depends, to a large extent, upon the cellular content of the tissue and is distinct 
from the phenomenon of coagulation. 

Many methods have been advanced for the determination of this property 
of the blood, that of Determann 1 being as clinically accurate and simple as any. 
So many uncertain factors influence the viscosity that the writer feels that this 
determination can add little to our clinical knowledge. As Burton-Opitz 2 
says, " Although the results have been gratifying in so far as normal viscosity- 
values have been established for the human blood, it seems doubtful whether 
this method will ever be perfected in a way that small variations can be accu- 
rately and safely recorded. Nor do the limitations lie wholly in the method. 
One of the gravest obstacles, encountered in establishing slight differential 
values, exists in the variability of the viscosity itself." 

It has been shown that the degree of viscosity (n) is influenced by cold, with- 
drawal and application of heat, the former factors causing an increase, while 
the latter lowers it. Hirsch and Beck demonstrate that the lower the specific 
gravity of the blood the less marked is its viscosity. These results agree with 
those that indicate that the lower the specific gravity the lower the number 
of cellular elements. We may readily see, therefore, why the blood in anemia and 
leukemia shows such a slight tendency to become sticky or to form rouleaux. 
The researches of Rotky show that n varies between 5.02 and 5.52 under normal 
conditions (water being 1), is 1.69 in anemias, 3.34 to 5.58 in nephritis, 13.56 
in febrile states and 16.93 m cyanotic conditions. According to Hess, the 
normal viscosity of male blood ranges between 4.3 and 5.3, while that of 
female blood varies from 3.9 to 4.9. The normal relation 3 

Hemoglobin 

Tr . . = 17 to 21. 

Viscosity 

(F). Coagulation of the Blood. 

It is impossible in this place to discuss the physics and chemistry of the 
process of coagulation, more than to say that this phenomenon is due to the 
conversion of the fibrinogen of the plasma into fibrin. This change takes 

1 Munch, med. Wchnschr., 1907, LIV, 1130; Die Viskositat des menschlichen B lutes., 
Wiesbaden, 191 o. 

2 Jour. Am. Med. Assn., 1911, LVII, 353. 

3 Austrian, Bull. Johns Hopkins Hosp., 1911, XXII, 9. See, also, Matsuo, Deutsch. 
Arch. f. klin. Med., 1912, CVI, 433. 

26 



402 DIAGNOSTIC METHODS. 

place under the influence of a ferment, thrombase, which is present in the 
leucocytes in the form of prothrombase. This latter zymogen, through the 
influence of calcium compounds, is changed into thrombase, the active agent 
in bringing about coagulation. 

It is occasionally of clinical importance to know the time of coagulation 
of the blood under certain conditions, as one of the normal processes of this 
tissue, when outside the vessels, is coagulation. This process takes place 
normally in from two to eight minutes, depending on several factors, among 
which are the length of time the blood is in contact with the tissues, the depth of the 
incision, the pressure with which the blood is expelled, the nature of the vessel 
into which the blood flows, and the temperature at which coagulation takes place. 

It has been found that certain variations in the normal coagulability are 
present at different hours of the day and are observed when blood is drawn 
from different parts of the body. 1 

Dorrance's Method. 

This method 2 is simple and reliable. The instrument consists of an 8- 
ounce thermos bottle without the silver lining, an aluminium stand and two 
rubber stoppers, one of which has one hole for the passage of a thermometer, 
while the other bears four holes each of which is lined with a brass flange. Into 
each of these holes fits a glass rod 41/2 inches long and 8 mm. in diameter. The 
lower end of each rod is cone-shaped, the flat tip being 4 mm. in diameter. The 
other end is slightly bulbous to hold the rod in place. 

Fill the bottle with water at 98 F. to within an inch of the top. Insert the 
stopper with thermometer. Scrub the glass rods with soap and water and cleanse 
them with alcohol and ether. Substitute the stopper with rods for the other, so 
as to warm them to 98 F. Puncture the ear or finger and wipe away the first 
two or three drops of blood. Remove the stopper with rods, wipe the latter 
dry and touch the drop of blood with the tip of each rod in turn, getting as nearly 
as possible the same amount of blood on each. Note the time and replace the 
stopper in bottle. 

The end-point is determined in three ways: (1) At the end of two and one- 
half minutes push rod 1 down into the water. All the blood normally falls off 
the end and breaks up into a fine cloud. At intervals of one minute the other 
rods in turn are pushed into the water. Some blood falls off but the particles 
become coarser, the amount remaining on the tip becoming greater until with 
rod 4 most of the blood remains, if coagulation has occurred. If no coagulation 

1 See Cohen, Arch. Int. Med., 1911, VIII, 684 and 820; Howell, Am. Jour. Physiol., 191 1, 
XXIX, 187 ; Therap. Gaz., 1912, XXXVI, 95; Bordet and Delange, Ann. de l'lnst. Pasteur, 1912, 
XXVI, 657; Berl. klin. Wchnschr., 1914, LI, 497; Barratt, Jour. Path, and Bacteriol., 1913, 
XVII, 303; Whipple, Arch. Int. Med., 1913, XII, 637; Cannon and Gray, Am. Jour. Physiol., 
1914, XXXIV, 232; Cannon and Mendenhall, Ibid., 225, 243 and 251; Gray and Lunt, Ibid., 
332; Howell, Ibid., 1914, XXXV, 143 and 474; Pepper and Krumbhaar, Jour. Infect. Dis., 
1914, XIV, 476; Fingerhut and Wintz, Munch, med. Wchnschr., 1914, LXI, 363; Stuber and 
Heim, Ibid., 1661; Fonio, Mitt. a. d. Grenzgeb. d. Med. u. Chir., 1914, XXVII, 642; Ibid., 
1914, XXVIII, 313; Lee and Vincent, Arch. Int. Med., 1914, XIII, 398; Ibid., 1915, XVI, 
59; Hurwitz and Drinker, Jour. Exper. Med., 1915, XXI, 401; Drinker, K. R. and C. K., Am. 
Jour. Physiol., 1915, XXXVI, 305; Mendenhall, Ibid., 1915, XXXVIII, 53; Denny and Minot, 
Ibid., 233; Bloch, Arch. d. Mai. du Cceur, 1915, VIII, 249. 

2 Am. Jour. Med. Sc, 1913, CXLVI, 562. 



THE BLOOD. 403 

obtains, the test is repeated with larger intervals between the immersions in 
water. (2) Remove stopper with rods and hold up to the light. The tips will 
show more or less of a red color depending on the degree of coagulation, rod 1 
being clear while rod 4 is almost covered with a red clot. (3) Touch the tip 
of each rod to filter paper. The depth of color of the blot will indicate the degree 
of clotting. The average coagulation-time by this method is three and one- 
half to five and one-half minutes. 

Rudolfs Method. 

This 1 is a modification of the older one of Sabrazes 2 and applies some of the 
points of the method of Kottmann. 3 It is so simple and clinically reliable that 
the writer advocates it for general use. Thin glass tubes 1.5 mm. in diameter 
and about 18 cm. (7 inches) in length are used. A pint Thermos bottle is 
employed, its ordinary cork being replaced by a triply perforated rubber stopper. 
In these perforations rest two brass tubes 7 inches long and just large enough in 
caliber to hold easily the glass blood-tubes. The third perforation contains a 
thermometer. The apparatus rests on its side in such a way as to prevent its 
rolling about. The bottle is filled with water at 20 C. and the stopper is 
inserted. 

The finger or lobe of the ear is punctured, the exact time of puncture being 
noted. Two glass tubes are partially filled from the same drop, the blood being 
made to run nearly to the far end of each tube, which is placed, as filled, in a 
brass tube of the thermostat. The protruding ends, the ones at which the 
blood entered the tubes, are then sealed with a spirit lamp. In about five 
minutes the first tube filled is drawn out of its holder by the left hand (covered 
with a glove to diminish the effect of heat of the fingers), is touched with a sharp 
file, broken across and the broken ends slowly separated. The tube is at once 
replaced in the thermostat. This technic is carried out with the first tube at 
intervals of 15 to 30 seconds, until a thread of fibrin appears between the 
broken ends. The time between the puncture and the appearance of the 
fibrin is the "coagulation time" of the blood. Tube No. 2 may then be used 
as a control. Normally the coagulation time averages eight and one-half 
minutes by this method. 

Method of Boggs. 

This is a modification of the older method of Russell and Brodie and gives 
a lower coagulation time than does the preceding method. It is based on the 
fact that the corpuscles, set in motion by a current of air in a moist chamber, 
move freely and independently of one another at first but, as coagulation pro- 
ceeds, clumping of the cells occurs, and finally an elastic radial motion of these 
cells obtains. This is observed under the low power of the microscope. The 
writer refers elsewhere for the technic (see cut) . 

Normally, the coagulation time, as evidenced by the formation of fibrin, 
is between two and nine minutes. 4 Anything above nine minutes means 

1 Am. Jour. Med. Sc, 1910, CXL, 807; 1911, CXLII, 481. 

2 Folia Hsemat., 1904, I, 394. 
3 Ztschr. f. klin. Med., 1910, LXIX, 415. 

4 Carpenter and Gittings (Am. Jour. Dis. Child., 19 13, V, 1) show that the average time 
for children is slightly longer than for adults. 



404 



DIAGNOSTIC METHODS. 



delayed coagulation. The importance of testing this function of the blood is 
observed more particularly in cases of suspected hemorrhagic diathesis, in 
which the period of coagulation is remarkably increased. In some cases 
of hemophilia it requires fifty minutes, while in certain of the purpuras from 
10 to 15 or more (Emerson). Likewise, it is customary to test this factor 
in cases of long-standing jaundice, in which surgical intervention for obstructive 
lesions of the biliary passages is to be undertaken, as here also the time is 
much increased. Moreover, we find the coagulation time delayed or imperfect 
in cases of hemoglobinemia, asphyxia, and general dropsy. 1 Poisoning from 
the bite of certain serpents, such as the cobra, is characterized by a greatly 
delayed coagulation of the blood as well as by hemolysis. On the other hand, 
we notice a quicker coagulation in conditions associated with stasis, repeated 
hemorrhages, transfusion, hunger, and also under the therapeutic use of 
calcium chlorid and of gelatin. 





Xdthar.rxe H.'l 



Fig. 122. — Boggs' coagulometer: a, moist chamber; b, tip of tube through which air 
passes; c, cover which fits over moist chamber and which holds glass cone; d, pin-hole for 
escape of air; ef, cross section of cover c; g, tip of glass cone upon which is placed the 
drop of blood. 

The formation of fibrin, as the end-product of coagulation, is increased 
in some cases and diminished in others. 2 An increase in the amount of fibrin 
in the blood (hyperinosis) is observed in acute inflammatory processes and in 
most infectious diseases. Hayem states that the density of the fibrin network, 
observed when blood is allowed to dry in thick smears on a glass slide, indicates 
the degree of resisting power of the individual against disease. The largest 
amounts of fibrin are observed in such conditions as pneumonia 3 and acute 
articular rheumatism, but are seen to a lesser extent in parenchymatous inflam- 
mation, in inflammations of the mucous membranes and the skin, in the 
febrile stages of chronic suppurations, in hepatitis, influenza, diphtheria, acute 
gout, and erysipelas. A decrease in the amount of fibrin (hypinosis) may be 
observed in malignant growths, malarial fever, pernicious anemia, leukemia, 
and purpura. In parenchymatous nephritis the amount of fibrin is but slightly 

1 See Kreiss, Inaug. Dissert, Heidelberg, 1912; Nel, Inaug. Dissert., Berlin, 1912; Lands- 
berg, Biochem. Ztschr., 1913, L, 245; Lee and White, Am. Jour. Med. Sc, 1913, CXLV, 
495; Jaffe, Folia Haemat, 1913, XV, 167; Steiger, Wien. klin. Wchnschr., 1913, XXVI, 1749- 

2 See Bosworth, Jour. Biol. Chem., 1915, XX, 91. 

3 Dochez (Jour. Exper. Med., 1912, XVI, 693) finds the coagulation time lengthened in the 
acute stage of pneumonia. 



THE BLOOD. 



405 



if at all increased, while in interstitial nephritis the increase may be notable 
(Da Costa). 

(G). Osmotic Pressure and Cryoscopy. 

The cryoscopic examination of the blood may be of some importance in 
the diagnosis of certain conditions, particularly in reference to the sufficiency 
or insufficiency of the kidneys as regards the elimination of the urinary solids. 
While this subject has not yielded as much information as was expected of it, 
a brief discussion seems essential. For a theoretical discussion of osmotic 
pressure and cryoscopy, I must refer the reader to 
works on physical chemistry. 

By cryoscopy from the Greek kryos, frost, and 
skopeo, to see, is meant the determination of the 
freezing-point of a solution and the referring of this 
figure to the freezing-point of the solvent, which is 
regarded as o. Substances in solution lower the 
freezing-point of the solvent in direct proportion 
to their molecular (dissociated or nondissociated) 
concentration. In determining the osmotic pressure 
of a solution, and cryoscopy is one of such methods, 
it is important to remember that the proteins have 
practically no osmotic value. We have, therefore, 
in this cryoscopic method a means of ascertaining 
directly the molecular concentration of the body 
fluids. 

The determination of the freezing-point of the 
blood is best made by means of the Beckmann ap- 
paratus, which may be found in works on physical 
chemistry. As the osmotic pressure of the serum is 
equal to that of the plasma or of the whole blood, 
the serum is generally used for this determination. 
Withdraw from a vein, preferably the median basilic, 
about 30 c.c. of blood and allow it to coagulate in a 
clean closed vessel. Place the serum in the freezing 
tube of the Beckmann apparatus, adjust the ther- 
mometer and stirring rods, and proceed as directed 
elsewhere. 

The freezing-point of the blood is usually desig- 
nated as the small Greek delta (5), while that of the 
urine is given the sign /^. Normally, blood freezes 
at — 0.56 C., distilled water freezing at o. This point is subject to more or 
less physiologic variation depending upon the changes which occur in the 
blood after meals, exercise, baths, etc. This physiologic variation is transient 
and slight because the kidneys soon regulate the osmotic pressure (molecular 
concentration) of the blood by withdrawal of the constituents which have 
caused the abnormal tension. This close relationship between the kidneys 




Fig. 123. — B eckmann 
apparatus. (Long.) 



406 DIAGNOSTIC METHODS. 

and the blood is of great importance in the study of renal insufficiency, as 
many products of metabolic activity remain in the blood in cases of renal 
disturbance. This is more particularly true of the inorganic constitutents of 
the blood, as the organic elements do not materially affect its osmotic pro- 
portions. It is in those cases of renal insufficiency with a tendency to uremia 
that the most is to be expected from the cryoscopic examination of the blood, 
yet we find that Schoenborn, 1 in the study of 88 cases, observed practically 
normal figures for the cryoscopic point of the blood. Engelmann, 2 od the other 
hand, reported a series of 36 cases in which the freezing-point averaged — 0.664 
C. It is true that in certain surgical conditions of the kidneys a parallelism is 
noticed between the lowering of the freezing-point and the development of the 
uremic symptoms. In cyanotic conditions of any origin whatever we find the 
cryoscopic point lower than the normal, owing to the fact that the C0 2 is in- 
creased in amount in such cases. If the C0 2 be driven off, the freezing-point 
becomes normal. Hence we see why, in certain cases of uncompensated cardiac 
disease, the freezing-point has frequently been reported much lower than the 
normal standard. There is practically no disease of the blood itself about 
which a cryoscopic study will furnish any information. 

From these considerations, as well as from many others, which I have 
not enumerated, the study of the cryoscopy of the blood does not seem to the 
writer of sufficient importance to warrant its adoption as a routine method of 
blood examination. It is, certainly, of no value whatever to the general 
practitioner who does not have access to a thoroughly equipped laboratory. 
The time consumed in such examinations would be much better spent in a 
more thorough study of the fresh and of the stained specimens. While 
cryoscopy may give some idea of the osmotic activity of the kidneys, 
it adds to blood examinations a slightly enlarged laboratory record, whose 
interpretation is a matter of more or less difficulty and whose results rarely 
ever give any information, beyond that which may be more easily and more 
readily learned by other methods. It is a purely theoretical method of follow- 
ing the metabolic activity of the system and reveals no signs of an oncoming 
uremia nor does it show us anything regarding the results of therapeutic 
measures. 

Naturally, variations in the osmotic pressure of the blood are of importance 
in the study of conditions associated with effusions into the various serous 
cavities, as such exudations are markedly influenced by the molecular con- 
centration of the blood. For a discussion of such phases of the subject I 
must refer the reader to works on pathology. 

(H). Electric Conductivity. 

In the effort to enlarge the scope of blood examinations, the clinician and, 
more particularly, the laboratory worker have taken advantage of everything 
offered for the furtherance of their aim. In so doing they have often over- 
stepped themselves and complicated the examinations by useless additions to 
their technic. 

1 Wiesbaden, 1904. 

2 Mitt., a. d. Grenzg. d. Med. u. Chir., 1903, XII, 396. 



THE BLOOD. 407 

By electric conductivity is meant the reciprocal of the resistance, which 
a certain amount of solution offers to the passage of an electric current of 
known strength between two platinum electrodes of given size and a given 
distance apart. This method is merely a measure of the number of electrolytes 
(both dissociated and nondissociated) in solution and is not affected by the 
nonelectrolytic organic substances of the blood, although, according to Hardy, 1 
the proteins move to one or the other pole of the battery depending on the 
reaction of the fluid. The method usually adopted for estimating this factor 
is that of Kohlrausch, the resistance being balanced on a Wheatstone bridge 
against a rheostat, the point of equilibrium being determined by means of a 
telephone attachment. 

As this determination can give us no information regarding the retention 
of organic products in the system, it is of comparatively little value in the 
study of metabolic or of blood diseases. It is in those conditions which are 
associated with retention of inorganic constituents that this method should be 
of value, and yet we find, according to the work of various authors, that in 
advanced nephritis, in which the retention of chlorids has been assumed, 
the conductivity of the serum is not increased to any extent, although the freez- 
ing-point is lower than the normal standards. This proves, its seems to the 
writer, that in such cases the substances capable of increasing the conductivity — 
that is, the chlorids — are either not retained in the blood or are in such a com- 
bination as not to influence the conductivity of this tissue. We must, therefore, 
assume that the lowering of the freezing-point observed in such conditions is 
due more to the organic than to the inorganic constituents of the blood. As 
this determination is, in the writer's opinion, of so little practical value in 
blood work, a detailed description of the methods and results of observations 
will not be taken up. 

(6). Chemical Properties. 

The chemical examination of the blood resolves itself into a consideration 
of the composition of the whole blood, of the plasma, of the serum, and of 
the various cellular elements. It is evident that the composition of the whole 
blood will depend upon the relations in which the single constituents of the 
blood stand to one another. 2 Naturally, physiologic influences are of great 
importance in the consideration of the composition of the fluid portion, while 
the cellular elements are less affected by these changes. In view of the recent 
work on opsonins and immunity, we must conclude that the chemical examina- 
tion of the fluid elements of the blood may yield much important information 
as soon as proper methods of research are evolved. 

Normally, the composition of the cellular elements remains almost con- 
stant and is but slightly affected by the varying composition of the fluid portions. 
As is well known, the corpuscles act more or less like semipermeable mem- 
branes, although Kahlenberg would have us believe that no such a condition 
is possible. At any rate, the membrane surrounding the erythrocyte allows 

1 Proc. Royal Soc, 1900, LXVI, 95. 

2 See Bonninger, Ztschr. f. exp. Path. u. Therap., 1912, XI, 1; also, Veil, Deutsch. Arch. f. 
klin. Med., 1914, CXIII, 226; Lowy, Deutsch. Arch. f. klin. Med., 1915, CXVII, 79. 



408 DIAGNOSTIC METHODS. 

the passage of certain inorganic and organic constituents of the plasma into 
the cells and permits the back-passage of certain constituents of the cells 
into the plasma. This question is intimately related to the subject of hemolysis, 
which will be discussed in detail later. It is true that a many-sided exchange 
exists between the red cells and the hypertonic plasma in which they float, 
such an exchange accounting very often for the alterations in the volume 
of the corpuscles and frequently being the cause of abnormally developed 
cells. This exchange is subject to certain restrictions. Thus we find that 
the corpuscles show a very high content in potassium salts and a very low 
sodium content, while the reverse conditions obtain in the serum. If free 
exchange took place between the cells and serum no such conditions could 
exist. It is known that potassium salts are closely associated with phenomena 
of growth and hence it might be possible to prove that cells, which are mor- 
phologically the older, show a less potassium content than do the newly develop- 
ing ones. 

It is evident that a quantitative analysis of the blood can be of only com- 
parative value as far as the blood as a whole is concerned. We should ascertain 
on one side the relationship of the plasma and blood-corpuscles to each other, 
and on the other side the composition of each of these two chief constituents. 
As there are many difficulties in the way of such determinations, we will not 
go into detail regarding the chemical composition of the different portions 
of the blood, but will discuss the blood as a whole. According to C. Schmidt, 
the composition of the blood is as outlined in the following table. This table, 
while giving the composition of the whole blood, may readily serve as one 
from which the composition of the corpuscles and serum may be obtained 
by reducing the constituents to parts per 1,000 of either corpuscles or serum. 
It is beyond the scope of this work to discuss the chemical properties of the 
blood or of its constituents in detail, but certain points which have clinical 
interest must be taken up. 

Man's. Woman's. 

Corpuscles 513-02 396.24 

Water 349.690 272.560 

Solids 163.330 123.680 

Hemoglobin, proteins, and 

other organic bodies .... 159.590 120.130 

Inorganic bodies 3-74° 3-55° 

K^O 1.586 1.412 

Na 2 0.241 0.648 

CaO ] 

MgO . . > 0.320 0.485 

Fe 2 3 J 

CI 0.898 0.362 

P2O5 0.695 °-643 



THE BLOOD. 409 

Man's. Woman's. 

Serum 486.98 603.76 

Water 439.020 551.990 

Solids 47.960 5i-77o 

Protein and other organic bodies 43.820 46.700 

Inorganic bodies 4.140 5-°7° 

K 2 o-i53 0.200 

Na 2 1. 661 1. 916 

CaO 

MgO 

CI 1.722 0.144 

P 2 O s 0.071 2.202 



0.533 °- 6 ° 8 



The large excess of chlorin in the serum of man as compared with that 
of woman and the excess of phosphoric acid in the serum of woman as com- 
pared with that of man is noteworthy. These variations may later be shown 
to have a great influence upon sexual differences as regards metabolism. 

(A). Total Solids. 

The determination of the total solids and, hence, of the water content 
of the blood is often of importance, especially in cases of anemia. 1 The method 
is as follows: Allow about i c.c. of blood to flow onto one of two previously 
weighed matched watch-glasses. Cover this with its mate and weigh them 
while in the moist condition. Separate the glasses and place them in a des- 
iccator over CaCl 2 or H 2 S0 4 for twenty-four hours, at the end of which time 
weigh them as before. The blood should have dried to a hard glassy mass 
in this time. The loss of weight will represent the water of the blood taken, 
from which amount the percentage may be calculated. Gumprecht and 
Stintzing and Biernacki advise the use of higher than room temperature, the 
former using 67 C. in an incubator, while the latter employs a heat of ioo 
to 120 C. Drying by these latter methods is often associated with loss; 
hence the writer is accustomed to use room temperature (about 20 C). 

Working in this way, many have found the dry residue (total solids) 
of the blood to be from 21 to 22.5 per cent., the water content being 77.5 to 
79 per cent. This figure shows marked variation under the influence of such 
processes as diarrhea, excessive sweating, and exudation into serous cavities. 
In anemia the solids are much reduced, while in leukemia they are increased. 

(B). Blood Pigments. 
Hemoglobin. 

The normal color of the blood is due to the presence of the pigments, 
hemoglobin and oxyhemoglobin. Usually the former is not large in amount, 
while the latter is the predominant factor in the color. In blood after asphyxia 
we find a mixture of hemoglobin, pseudohemoglobin, and parhemoglobin; 
in arterial blood large amounts of oxyhemoglobin, and in venous blood a 
mixture of hemoglobin and oxyhemoglobin. 

1 See Busch, Ztschr. f. exper. Path. u. Therap., 1913, XIV, 335. 



410 



DIAGNOSTIC METHODS. 



Hemoglobin belongs to the class of bodies known as "chromo proteins'' 
and, owing to its power of combining with various gases and thus aiding in 
the gaseous exchange of the body, may be styled a "respiratory protein." 
It is easily decomposed into a protein, globin (about 96 per cent.), and a pig- 
ment, hemochromogen (4 per cent.), which contains iron and is easily changed 
in the presence of oxygen into hematin. The iron content of hemoglobin 
is the portion which enables this pigment to exert its peculiar vital power of 
oxygen transference. 

The amount of hemoglobin in normal blood is variable, depending on 
the age of the subject examined. Normally, 100 c.c. of adult human blood 
contain from 13 to 14 grams of hemoglobin (the average being about 13.77), 
which amount is not a constant factor at all ages. From the following table 
of Leichtenstern it will be seen that a definite age curve exists for this substance. 

Age. Grams per 100 ex. of blood. 

1 to 4 days, *9-3 2 9 t0 21.160 

8 to 14 days, 17.869 to 16.124 

8 to 20 weeks, 15-362 to 12.928 

6 months to 5 years, 10.971 to 11.373 

5 to 15 years, n.15 1 t0 n-79 6 

15 to 25 years, i3-°34 to 13.870 

25 to 45 years, i4-7 2 7 t0 I 5- OI 3 

45 to 60 years, 12.484 to 13.150 

Over 60 years, 14-79° 

Under certain conditions the hemoglobin is dissolved out from the red 
cells, leaving only their stromata behind. This gives rise to the condition 
known as hemoglobinemia which will be discussed in a later section. Accord- 
ing as hemoglobin is free or combined with certain gases, we have in the blood 
various derivatives of this pigment, each one of which has certain characteristics 
and a definite clinical importance. These derivatives are recognized by 



Ar 



iiiiiaw 



Fig. 124. — Direct- vision spectroscope. 



qualitative tests, especially by spectroscopic methods, and will be presented 
here; while the important tests used for the recognition of hemoglobin, either 
in the fresh state or as dried stains, will be taken up under the medicolegal 
discussion. 1 

Hemoglobin, also called reduced hemoglobin, is much more soluble than 

oxyhemoglobin, its solution in water being more violet or purplish than one 

of oxyhemoglobin of the same concentration. Such solutions of hemoglobin 

absorb the blue and violet rays of the spectrum to a less marked degree than 

1 See Maestre and Lecha-Marzo, Arch, internat. de med. leg., 1914-j V, 49. 



3B % 



Absorption Spectra. 

PLATE XV. 

2) 



^r 



& 



1 










j 




























! 












1 



Oxyhaemoglobin. 



Haemoglobin. 



Carboxy- 

haemoglobin. 



Neutral Met- 

haemoglobin. 



Alkaline Met- 

haemoglobin. 



Alkali 
Haematin. 



(FROM HAWKS "PHyS/OLOG/CAL CHEMfSTRr") 



Absorption Spectra. 



a % 



PLATE XVI. 

3> £ ^ &~ 



10 



12 



13 



14 





















1 








1 


[ 












































. 




1 1 



Reduced Alkali 
Haematin or 
Haemochromogen. 



Acid Haematin in 
ethereal solution. 



Acid Haemato- 
porphyrin. 



Alkaline 

Haematopor- 

phyrln. 



Urobilin or Hydro- 
bilirubin in acid 
solution. 



Urobilin or Hydro- 
bilirubin-in alkaline 
solution after the 
addition of zinc 
chloride solution. 



Bilicyanin or 
Cholecyanin in 
alkaline solution. 



(FROM HAWKS "f>HYS/OLOG/CAL CHEMISTBr") 



THE BLOOD. 411 

do those of oxyhemoglobin, but they strongly absorb the rays lying between 
C and D. In proper dilution a solution of hemoglobin shows a spectrum 
with one broad not clearly denned band between D and E, lying toward the 
red end of the spectrum a little over the Fraunhofer line D (see plate). 

Pseudohemoglobin. 

According to Ludwig and Siegfried, blood, reduced by hyposulphites 
or by a stream of hydrogen to such an extent that the spectrum of oxyhemoglobin 
disappears, yields large amounts of oxygen when exposed to a vacuum. This 
loose combination of hemoglobin and oxygen, which gives the spectrum of 
hemoglobin, is called pseudohemoglobin. Hammarsten considers it an in- 
termediate body between hemoglobin and oxyhemoglobin. 

Oxyhemoglobin. 

Oxyhemoglobin, also called hematoglobulin, is a molecular combination 
of hemoglobin and oxygen. The ability of hemoglobin to take up oxygen 
is a function of its iron content. When this factor is calculated as 0.33-0.40 
per cent., 1 atom of iron in the hemoglobin molecule corresponds to two atoms 
(one molecule) of oxygen. This combination is a loose one and hence the 
quantity taken up will depend upon the partial pressure of the oxygen. This 
oxygen is set free when the oxygen pressure is reduced, thus giving rise to the 
characteristic property of oxygen transference. As Pfliiger has shown, oxy- 
hemoglobin may, when it is gradually oxidized, act as an " ozone exciter" 
by the decomposition of neutral oxygen into the atomic form. It may also 
act as an " ozone transmitter" as in certain tests to be outlined later. 1 

A dilute solution of oxyhemoglobin or of arterial blood shows a spectrum 
with two absorption bands between the Fraunhofer lines D and E. One 
band, «, is narrower, but darker and sharper and lies on the line D, the other, 
p, is broader, less denned and less dark and lies at E. As the dilution becomes 
weaker the band /? first disappears. By increased concentration the two 
bands become broader, the space between them smaller, and the blue and 
violet parts of the spectrum darkened. Other substances may give this same 
absorption spectrum, but oxyhemoglobin may be differentiated by its be- 
havior toward reducing agents, such as ammonium sulphid or Stokes' solution 
of ammoniacal ferrotartrate (see plate). 

Methemoglobin. 

This pigment is closely related to oxyhemoglobin, as it contains the same 
amount of oxygen and is isomeric with it. The oxygen is, however, not in 
loose combination and cannot, therefore, be utilized by the system. This 
coloring matter is formed by the spontaneous decomposition of blood, as 
observed in hemorrhagic transudates and cystic fluids, and occurs also 
in cases of poisoning with potassium permanganate, potassium ferricyanid, 
chlorates, nitrites, nitrobenzol, acetanilid, antipyrin, turpentine, sulphonal, 
and arsenic, and in cases of cyanosis with diarrhea. 

1 See McClendon, Jour. Biol. Chem., 1915, XXI, 275. 



412 DIAGNOSTIC METHODS. 

According to Jaderholm and Bertin-Sans, the absorption spectrum of 
methemoglobin, in aqueous or slightly acidified solution, is similar to that of 
acid hematin (see below), but is easily distinguished from the latter by the 
readiness with which it turns into that of hemoglobin on treatment with alkali 
and a reducing substance. Hematin, under the same conditions, gives the 
spectrum of an alkaline solution of hemochromogen (Hammarsten). Met- 
hemoglobin, in alkaline solution, shows two absorption bands, which are like 
those of oxyhemoglobin, but differ from them in that the band nearer E is 
stronger than the one at D. A third fainter band may be observed, according 
to Hammarsten, lying between C and D. 

Carbon-monoxid Hemoglobin. 

This pigment is a molecular combination of one molecule of hemoglobin 
with one molecule of CO. This combination is stronger than those of hemo- 
globin and oxygen. The oxygen of hemoglobin is easily replaced, therefore, 
by CO and, in consequence, the tissues suffer for want of oxygen. This pig- 
ment imparts to the blood a bright cherry-red color both in the venous and 
arterial circulation. 

The most common cause of formation of this pigment is the inhalation of 
coal gas or of illuminating gas. The characteristic color in these cases may 
disappear after a few hours or it may persist for days, depending on the severity 
of the case, being frequently found in the blood after death. Such blood 
shows an absorption spectrum similar to that of oxyhemoglobin, but the two 
bands lie more toward the violet end of the spectrum than in the case of oxy- 
hemoglobin. On the addition of a reducing agent to blood showing this 
spectrum, the absorption bands of carbon-monoxid hemoglobin are unaffected, 
while those of oxyhemoglobin are changed to those of hemoglobin. This 
spectroscopic test is not delicate as less than 20 per cent, of carbon monoxid 
hemoglobin in the blood is difficultly, if at all, detected. 

Carbon-dioxid Hemoglobin. 

According to Bohr, hemoglobin forms three molecular combinations with 
C0 2 , in which products, <*, £, and 7, one gram of hemoglobin combines with 
1.5, 3, and 6 c.c. of C0 2 . The spectrum of these compounds is similar to 
that of reduced hemoglobin. If a large excess of C0 2 be present the hemoglo- 
bin is decomposed and globin is precipitated. The absorption band under 
these conditions is probably referable to the presence of free hemochromogen. 

Sulph-hemoglobin. 

This pigment, formed by the action of hydrogen sulphid upon hemoglobin, 
is found in the corpuscles and not free in the plasma. Sulph-hemoglobinemia 
has been reported by several, especially by van der Bergh 1 whose name is given 
to the condition known as "idiopathic enterogenous cyanosis" and character- 
ized by cyanosis, headache and marked constipation. Clarke 2 has reported 

1 Deutsch. Arch. f.klin.Med., 1905, LXXXIII, 86; Berl. klin. Wchnschr., 1906, XLIII, 7. 

2 Medical Record, 1909, LXXVI, 143; 1910, LXXVIII, 987. See, also, Wallis, Quart. 
Jour. Med., 1913, VII, 73. 



THE BLOOD. 



/ 

413 



the first case in the United States. Spectroscopic examination of the diluted 
blood is essential to show the presence of this pigment. The spectrum shows 
3 absorption bands, two similar to those of oxy-hemoglobin and a third in the 
red not so near the C line as is the third band of methemoglobin. Addition of 
dilute ammonium sulphid destroys the third methemoglobin band, while it does 
not effect that due to sulph-hemoglobin. 

Decomposition Products of Blood Pigments. 

As stated previously, hemoglobin and oxyhemoglobin are proteins, which 
are converted under the action of different physical and chemical means into 
a globulin-like protein, globin, and an iron-containing pigment, hemochromo- 
gen. From the table below, adapted from Webster and Koch, 1 may be seen 
the relations of hemoglobin to its various derivatives and also to the bile 
pigments and to chlorophyll. 



(C 32 H 50 N 4 FeO 3 HCl) 
Hematin (C 32 H 31 3 N 4 Fe)— >Hematoporphyrin (Cj 6 H 18 3 N 2 ) 

/ II 

in/ Hemochromogen / Phylloporphyrin (C 16 H 18 N 2 0) 

Hemopyrrol (C 8 Hi 3 N)— Phyllocyanin«-Chlorophyll 



Hemin 

it. 



Hemoglobin/ Hemochromogen 




Urobilin (C 32 H 40 O 7 N 4 ) 



Bilirubin (C le H I8 N 2 3 )± 



^.Hematoidin 



Biliverdin (C 16 H 18 N 3 4 ) 



Hematin. 

This decomposition product is found in any situation in which oxyhemo- 
globin is destroyed; thus, in the digestive tract where it is formed by the action 
of gastric or pancreatic juice on oxyhemoglobin, in old extravasations, in the 
stools after hemorrhage, in urine after poisoning with arsenical compounds, 



laboratory Manual of Physiological Chemistry, Chicago, 1903. 



414 DIAGNOSTIC METHODS. 

and in the blood of persons poisoned with nitrobenzol and acetanilid. 1 Hema- 
tin is a dark brown or blue-black amorphous powder, insoluble in water, 
dilute acids, alcohol, ether, and chloroform, but readily soluble in acidified 
alcohol or ether and in dilute alkalies. 

Arnold has shown that there are three modifications of this pigment, a 
neutral, an alkaline, and an acid hematin, each showing spectroscopic differ- 
ences. The neutral hematin has little importance clinically and will be passed 
in this discussion. Acid-hematin solutions show four absorption bands, one 
between C and D, a second broad but not clearly defined band between D and 
F, which divides under certain conditions into two narrower bands, a fourth band 
between D and E which is nearer D and is very weak. Usually only the band 
between C and D and the broad band between D and F can be seen (see plate). 

Solutions of alkaline hematin show one absorption band between C and 
D which reaches out to some extent between D and E. If the alkaline hema- 
tin solution be reduced with ammonium sulphid, the spectrum of hemo- 
chromogen is observed, which shows two characteristic absorption bands, 
one very sharp and dark between D and E and a second paler and broader 
band covering the E line. 

Hematin forms very characteristic compounds with hydrochloric, hydro- 
bromic, or hydriodic acids. With HC1 hematin crystallizes with one mole- 
cule to form the compound hemin. The crystals are light or dark brown 
rhombic forms and are called, after their discoverer, Teichmann's crystals. 
Their formation is specific for blood, but the kind of blood cannot be deter- 
mined by their presence. As their detection is largely a medicolegal question, 
I will refer a discussion of the technic to the later section. 

Hematoporphy rin . 

When hematin is treated with concentrated sulphuric acid in preseDce of 
air, iron is split off leaving the pigment hematoporphyrin. If air be excluded 
the product yielded by such treatment is hematolin. 

This pigment is insoluble in water, but dissolves in alcohol, strong and 
weak alkalies, and in acids. It is isomeric with bilirubin, with which it is 
associated in the liver cells. In acid solution hematoporphyrin shows two 
absorption bands, one fainter and narrower between C and D and nearer 
D, the other darker, sharper, and broader in the middle between D and E. In 
dilute alkaline solutions this pigment shows four absorption bands. A band 
between C and D, a second broader band surrounding D with the broadest 
part between D and E, a third between D and E nearly at E, and a fourth 
broad and dark band between E and F. Hematoporphyrin is of great impor- 
tance from the medicolegal standpoint as certain suspected stains may be 
identified only by its spectrum. For this phase see the later sections. 

Hematoidin. 

This ruby-red or reddish-yellow pigment is derived from blood coloring 
matter and like hematoporphyrin is iron free. It is found in old blood-clots, 

1 Schumm (Ztschr. f. physiol. Chem., 1912. LXXX, 1) reports hematinemia in a case of 
acute chromium poisoning. 



THE BLOOD. 415 

in hemorrhagic exudates, in sputum, and in feces. It is more abundant 
when the blood pigment is not much exposed to the action of living cells, as 
in the center of large extravasations and in hemorrhages into preformed cavities 
of the body (Ziegler). Hematoidin is identical with bilirubin and shows no 
absorption bands, but only a strong absorption of the violet to the green portion 
of the spectrum. 

Hemosiderin. 

This yellow, orange, or brown pigment is a derivative of hemoglobin and 
contains iron. Unlike hematoidin, it is found more particularly in extrava- 
sated blood which has been subjected to the action of living cells. After a time 
this pigment changes into one (probably hematoidin) which contains no iron. 

Malarial Pigment. 

The older view of Ewing 1 and others that this yellowish-brown or black 
pigment was melanin seems to have been disproven by the later work of 
Brown 2 who has shown that it is, probably, identical with hematin and is 
formed by the action of the malarial parasite (possibly of a proteolytic enzyme) 
upon hemoglobin. On spectroscopic examination of an alkaline alcoholic 
solution of this pigment, a single broad band is seen starting sharply at D and 
extending to the left, gradually shading into the red between C and D. The 
melanins, observed in the leucocytes in relapsing fever, melanotic sarcoma and 
Addison's disease, are quite distinct from this malarial pigment as shown by 
decolorizing and solubility reactions. 

Estimation of Hemoglobin. 

A very large number of methods have been introduced for the deter- 
mination of the blood coloring matter. Some of these are extremely accurate, 
but are too complicated for general clinical purposes ; others are less accurate, 
but are more applicable to our work. It may be said that, as a rule, the clinical 
instruments at our command will give comparative figures, providing their 
construction and their standardization is accurate. However, we find so much 
inaccuracy or even lack of proper standardization in some of these instruments, 
that no absolute comparison may be attempted between the results obtained 
by various workers using the same or different methods. 

Normal adult blood contains about 13.77 grams of hemoglobin per 100 
c.c. of blood, this figure being subject, however, to variations at different 
periods of life, so that those instruments which are standardized against normal 
adult blood and which furnish the amount of hemoglobin in terms of per- 
centage of such blood cannot be absolutely accurate no matter how perfectly 
constructed or how accurately standardized, when they are used for the esti- 
mation of hemoglobin in the blood of a child or of an elderly patient. It is, 
therefore, much better practice to obtain the values in terms of the actual 
amount per 100 c.c. of blood. It is well to recognize, as Turk points out, 
that we are working with possible errors and can obtain comparative results 

1 Jour. Exper. Med., 1905, VI, 119. 

2 Arch. Int. Med., 1911, XIII, 290. 



416 DIAGNOSTIC METHODS. 

only when we use the same exactitude and care in each of our estimations. 
The errors are essentially constant, but we must remember that the arbitrary 
color standards change as time goes on, necessitating a restandardization 
of our instruments if accurate results are to follow. The writer has seen errors 
of 50 per cent, arise from the use of an old von Fleischl instrument and one 
of 25 per cent, from a faulty standardization of Sahli's solution. 

It must be insisted upon that we must not assume that a patient, who is 
pale and " anemic looking," is in reality anemic. Pallor depends not only 
upon the amount of pigment in the blood but also upon the delicacy and 
transparency of the skin and upon the superficial distribution and size of the 
blood-vessels. By remembering these points we may often save ourselves 
unnecessary chagrin on finding that the results of our determinations do not 
accord with the "anemic " expression of the patient. A simple puncture 
of the ear, allowing the blood to drop upon a clean linen towel (so-called 
"towel test"), will many times set us right and prevent a diagnosis of anemia 
without further examination of the blood. Such examinationless diagnoses 
are unwarranted and inexcusable. 

Direct Methods of Estimation. 

These methods are, owing to their complexity, not applicable to clinical 
work. The spectrophotometer of Hiifner is undoubtedly the very best and 
most accurate method, but I must refer to the original article for its description. 
Likewise the colorimetric double pipet of Hoppe-Seyler and the methods 
of Nebelthau and of Zangemeister must be neglected. 

Indirect Colorimetric Methods. 

These indirect methods employ a comparison of the blood solution with 
a second medium which approximates the blood in color. Two principles 
are possible in such methods. We may either have a fixed unchangeable 
medium of comparison, with which the blood is matched by constant dilution, 
or we may use a fixed blood solution of known strength and compare there- 
with the standard graduated color scale. Both of these principles have been 
used in such determinations, the variations being shown in the fact that instru- 
ments for this purpose have been introduced by Gowers, Sahli, Hayem, Malas- 
sez, Henocque, Bizzozero, von Fleischl, Miescher, Haldane, Griitzner, Gartner, 
Dare, Oliver, and Tallqvist. 

It is evident, a -priori, that these indirect methods must have greater 
errors than the direct. Two things are essential for any exactitude whatever. 
First, the medium of comparison must agree in the most complete manner 
with the various color tones which the blood shows at different percentage 
values of hemoglobin. Second, the standardization of the medium of com- 
parison must be extremely accurate. We must have no illusions regarding 
the exactness of the hemoglobin values obtained by these methods, as it is 
very difficult to construct two instruments of the same kind that will agree 
exactly. Certainly no two instruments of different make will agree, but com- 
parative results sufficiently accurate for clinical purposes are obtained by the 
use of the more reliable instruments. * The personal equation in reading the 



THE BLOOD. 



417 



color comparisons must be remembered, as some individuals show abnormal 
sensitiveness or lack of sensitiveness to shadings of red. It is an impossibility 
in this book to give in detail all of the methods advanced for the estimation of 
hemoglobin. I select, therefore, those that have proven most reliable in my hands. 

Hemometer of Fleischl-Miescher. 

Up to recent times the most frequently used of the instruments for the 
estimation of hemoglobin was the old von Fleischl instrument. With the 
introduction of the Miescher modification, this original form has been or should 
be less often employed. We avoid, therefore, a discussion of the older instru- 
ment, referring to other works which have included it. 

This new apparatus, made by Reichert, under the direction of Miescher, 




Fig. 125. — Hemometer of Fleischl-Miescher: R, Stage; T, milled head, which moves 
the color scale; m, opening in stage through which the instrument is read; M, mixing cell; 
D', cover glass; D, cap; PS, gypsum mirror from which light is reflected; mel, diluting 
pipet. 



is similar in general appearance to the old von Fleischl. It has the same stand 
and the same scale principle, although this latter is standardized differently and 
graduated on a different basis. It differs, materially, in the method of measur- 
ing and diluting the blood, in the form of the comparison chamber, and in the 
meaning of the graduation of the scale (see cut and legend for its description). 

The Diluting Pipet. 

This is similar in construction to the pipet of the Thoma-Zeiss hemo- 
cytometer, its calibrations, however, being different. The marks are 1/2, 
2/3, and 1. Above and below each of these main divisions are two marks 

27 



418 DIAGNOSTIC METHODS. 

each corresponding to i/ioo of the contents of the capillary tube. This 
device enables the worker to measure accurately the column of blood taken, 
in case he gets too little or too much blood in the tube. The relation 
of the capillary to the ampulla is such that blood, drawn to the mark i and 
diluted to the mark above the ampulla, receives a dilution of 200; if drawn 
to the mark 2/3 the dilution is 300; while the line 1/2 furnishes a dilution of 
400. The diluent used is 1/10 per cent, sodium carbonate solution. This 
dissolves the stromata of the red cells furnishing a clear solution. Occasionally 
the diluent becomes turbid after standing some time, and should be freshly 
made and should contain no bicarbonate. 

The taking of the blood and the mixing with a diluent is done with the 
same precautions mentioned under the method of making the blood count. 
In choosing the proper dilution, one should select that which will enable 
him to use the central portions of the graduated scale. A dilution of 400 is 
usually applicable except in cases in which marked anemia is suspected when 
dilutions of 300, 200, or even 100 should be made. In making this last dilu- 
* tion the erythrocytometer may be used. In doing accurate work, it is well to 
make a preliminary determination of hemoglobin in order to tell, the better, 
just what dilution would be advisable. This may be done by the Tallqvist 
method described later. 

The Comparison Cells. 

With this instrument two cells for holding the blood and diluent are 
furnished. One of these has a depth of 15 mm., the other one of 12 mm. 
the former is the standard cell, the latter the control, giving 4/5 the value of 
the larger cell. Their external appearance is similar to the cell of the old 
Fleischl, but their capacity is less owing to their greater thickness. 

The dividing partition between the halves of the cells projects about 
1/2 mm. above the borders, thus preventing any mixing of the fluids 
in the two portions. A grooved cover-glass is slid over the compartments 
without fear of mixing the fluids. If fluid is lost no error is introduced, as the 
dilution is uniform in the pipet and the depth of the chamber is definite. 
A diaphragm, with an opening 4 mm. wide, is placed over the cover-glass 
after this latter has been adjusted, the opening being so placed that its long 
axis is perpendicular to that of the vertical partition. The field of comparison 
is thus limited to 4 mm., which corresponds to about 3 of the scale, affording 
a comparison of a single tint of the scale with the color of the diluted blood. 

Graduation of the Scale. 

The slide for comparison of color is tinted with Cassius' golden-purple, 
as in the old Fleischl instrument. The graduations of this color scale are 
made by comparison with standard solutions of hemoglobin and not with the 
arbitrary standard of blood of so-called normal individuals. The scale shows 
the same divisions as that of the Fleischl instrument, but their significance 
is different in the Miescher modification. Here one does not read directly 
the percentage of hemoglobin, but must obtain the corresponding value by 



THE BLOOD. 419 

reference to a "table of calibrations," which accompanies each instrument. 
While the figure thus obtained is in terms of actual percentage (grams per 100 
c.c), it is quite different from the percentage figure given by the Fleischl 
instrument. 

Method. 

After testing the chamber by placing in one compartment the diluting 
fluid in order to see that none runs into the other, the blood is drawn into the 
pipet to the desired point and diluted to the mark, all precautions mentioned 
under Blood Counting being observed. Thoroughly mix the blood and diluent 
by shaking and blow out the unmixed contents of the capillary tube. Fill one com- 
partment of the 15 mm. cell with the diluted blood so that a convex meniscus 
appears above the border of the chamber, the other compartment being filled 
with the diluent or with distilled water. Adjust the grooved slide and cap, 
place the cell in the central opening of the stand, and adjust the light. This 
latter portion of the technic is of considerable moment, as this instrument 
gives the best results when used in a dark room illuminated only by a small 
candle flame which is placed about 18 inches from the stand and to the side. 
In the absence of a dark room, a light-proof shield for the eyes may serve. 
This may readily be obtained by the use of a tube of stiff, dark paper, which 
fits over the comparison cell and shuts off the light of the candle from the 
field of vision. The observer should stand in such a position that he looks 
into the cell from the side and not from the front or back, the eyes being about 
one foot above the cell. In the comparison of the color tones the variations 
are much better seen by quick movements of the slide rather than by slow 
gradual changes. The eyes should be rested at short intervals to prevent 
fatigue and lack of sensitiveness to the different shadings of color. When 
the color is matched take the reading of the instrument, by observing what 
mark of the scale coincides with the notch on the edge of the opening above 
the scale, and control this reading by several duplicates. 

Remove the blood from the 15 mm. cell by means of the pipet and 
transfer it to one compartment of the 12 mm. chamber, tested as was the larger 
one. Adjust this chamber and make readings in the same way. These 
values should be only four-fifths of those obtained with the 15 mm. cell. 
This modification has the advantage of using different portions of the color 
scale and should give comparative figures. If there should be any variation, 
which should never be over 1 per cent., a correction may be made. 

Calculation of Results. 

This is possible only with the use of the "table of calibrations," which 
contains the series of scale divisions and the absolute amount of hemoglobin 
in mg. per 1,000 c.c. of blood, corresponding to each division of the scale 
when the 15 mm. chamber is used. Thus, if the scale shows 56, we find the 
value corresponding to this to be 447 mg. of hemoglobin in 1,000 c.c. of diluted 
blood. As the dilution may have been 400, we would have, in 1,000 c.c. of 
undiluted blood, 400 x 447 = 178.8 grams. As we wish to know the amount 



420 



DIAGNOSTIC METHODS. 



in ioo c.c. we have merely to divide by 10, thus obtaining 17.88 grams of 
hemoglobin. If we wish to get the percentage figures, as read on the old 
Fleischl, we divide the figure obtained with the Miescher, in this case 17.88, 
by the amount of hemoglobin corresponding to the 100 division of the scale 
of the instrument used, in this case 14, and we obtain 127.7 P er cent - 

This Miescher modification is our very best clinical apparatus for estima- 
tion of hemoglobin, giving results which are accurate within 0.2 to 0.5 per 
cent. It is open to the objections that it is bulky, expensive, and requires 
more time and practice for its use than is at the control of the busy practitioner. 
For hospital use, however, it is the instrument par excellence and should never 
be substituted by others. It has one disadvantage that each instrument is 
standardized, not against a known hemoglobin solution, but against a so- 
called " normal" instrument, which 
has been properly standardized. It is 
to be hoped that each instrument will, 
in the future, be properly calibrated 
and thus insure us against the possi- 
bility of errors arising from any change 
in the standardization of the "normal" 
instrument. 

Hemoglobinometer of Dare. 

The instrument, introduced by 
Dare, has the advantage of using un- 
diluted blood, and avoids any error 
consequent upon dilution. The prin- 
ciple of this instrument is as follows: 
The color of undiluted blood is com- 
pared by artificial light with that of a 
graduated glass scale colored with 
golden-purple, the 100-point of which 

is standardized against a solution of 13.77 grams of hemoglobin in 100 c.c. 

of serum. For a description of the apparatus see cut. 




Fig. 126. — Hemoglobinometer of Dare: 
R, milled wheel; s, case inclosing the color 
disk; t, movable wing, which is swung out- 
ward; u, telescoping camera; v, aperture 
admitting light; w, capillary blood pipet; 
y, detachable candle holder; z, slot through 
which the percentage of hemoglobin is read. 



Method. 

Sw T ing outward the movable screen, which serves as a cover for the case, 
adjust the camera tube, and fit the candle attachment in its place opposite 
the camera tube. The candle should be so adjusted that its upper end is 
flush with the top of the clips which hold it. If the wick be curved, it should 
be so turned that the intensity of the light is midway between the two apertures. 
See that the pipet, composed of the rectangular glass plates, is thoroughly 
cleansed and dry. The space between the plates is filled by applying the edge 
of the pipet to the side of a fairly large drop of blood. Adjust this pipet 
in its place and rotate the colored scale, by means of the milled screw, until 
the colors match. Hold the instrument steady to prevent the flickering of the 
flame as much as possible. No dark room is necessary, but it is advisable to 



THE BLOOD. 42 1 

point the instrument at some dark object and to avoid direct sunlight, as the 
shadings of color are not so easily matched by direct daylight. As soon as 
the colors are matched make the readings and check the results with several 
duplicates. This reading is observed on the left side of the case in the small 
open space, the line which coincides with the beveled edge of the opening 
representing the percentage of hemoglobin, on the basis of a value of 13.77 
grams of hemoglobin per 100 c.c. as 100 per cent. It is, therefore, easy to 
calculate the direct amount of hemoglobin in the blood examined. 

This instrument has the advantages that undiluted blood is used, that the 
scale of comparison is usually very accurately standardized, that it is con- 
venient, easy of manipulation, and rapid in giving results. Coagulation of 
the blood does not occur sufficiently soon to introduce an error, providing the 
reading is taken within a reasonable time. It is more convenient for general 




Fig. 127. — Method of filling the Dare blood pipet. {Da Costa.) 

use than is the Miescher, is less expensive, can be used in a light room, and 
gives results second only to those of the Miescher. The disadvantages of this 
instrument are that an occasional faulty standardization may introduce errors, 
it costs much more than some of the instruments to be described, and it is 
not a long-lived instrument unless care is taken in handling it. In the writer's 
laboratory this instrument has given great satisfaction and can be recommended 
for general use on the ground of its convenience and ready application to clinical 
work. 

Hemometer of Sahli. 

This instrument is a new modification of the older hemoglobinometer 
of Gowers, and has so many advantages over the older instrument that this 
latter will be passed over. A modification of the Gowers instrument intro- 
duced by Haldane is simple and reliable, but has the disadvantage that coal gas 
is used in converting the hemoglobin into CO hemoglobin, and consequently, 
is not of easy application for bedside work. 

As Sahli has pointed out, a colored fluid under examination should not 
be compared with a different substance similar in color, but with a solution 
of known strength of the same coloring matter. His modification of the 
Gowers method employs an instrument constructed on exactly the same lines, 
but using a different standard of comparison. This standard of comparison 



422 



DIAGNOSTIC METHODS. 



is an acid hematin solution in a concentration corresponding to a i per cent, 
solution of normal blood. This solution is somewhat dark, as it is standardized 
against blood showing high hemoglobin value. As Sahli states, the standard 
fluid, as furnished with his hemometer, corresponds to a blood which shows 
with the Miescher instrument at a dilution of 200 a reading of 109, or an ab- 
solute quantity of 17.2 grams of hemoglobin per 100 c.c. of blood. The fine 
particles of hemin, which are in suspension, may 
adhere to the glass, especially if the instrument lies 
unused for some time. This process changes the color 
of the standard to some extent, so that it is supposed 
that deterioration has occurred. This may be remedied 
by completely inverting the tube, without violent shak- 
ing, thus allowing the precipitate to diffuse uniformly. 
Occasionally one of these instruments is found to be 
improperly standardized, but this is rare. 

Method. 

This consists in diluting the blood with 10 times 
its volume of N/10 normal hydrochloric acid. After 
a few seconds the fluid becomes dark brown from the 
formation of hematin hydrochlorid (hemin), which 
substance is not in solution, but in fine suspension. 
The blood is taken with the 20 cmm. pipet and 
blown into the graduated tube, which contains N/10 
normal HC1 up to the mark 10. This acid may be 
accurately enough made by diluting 15 c.c. of concen- 
trated chemically pure HC1 to 1,000 c.c. Shake the 
mixture of blood and acid and dilute with ordinary 
water as soon as a clear dark brown color is visible. 
Add water until the shade of the mixture corresponds 
exactly with that of the standard solution, when the 
percentage of hemoglobin may be read off. The 
water should be added very carefully from a dropping pipet, as the accuracy 
of the method depends upon adding the exact amount of fluid necessary. 
The comparison of colors may be made in any light, as the two solutions, 
being the same, will be similarly affected. 

This instrument is very conveniently gotten up, being mounted in a case, 
carrying a white-glass plate to reflect the light to better advantage. It is 
simple, inexpensive, and is accurate within 2 per cent. The author would 
recommend this instrument to the general worker above all others, with the 
possible exception of the Dare, whose advantages have been mentioned. 

Hemoglobinometer of Oliver. 

In this method the color of the blood in a definite dilution is compared, 
by light reflected from a white surface, with a series of tinted glass standards. 
Such a method has the advantage that the color of the diluted blood is com- 
pared with one single tint at one time. The standard glass disks correspond 




Fig. 128. — Hemometer of 
Sahli. {Greene.) 



THE BLOOD. 



423 



accurately, as determined by the tintometer, to the specific color curve of 
progressive dilutions of normal blood. Two sets of standards are furnished, 
one for use in daylight, the other by candlelight, the latter being preferable. 
Each set of 12 disks is mounted on plaster of Paris and enclosed in two 




Fig. 129. — Hemoglobinometer of Oliver. (Coplin.) 

wooden frames, six disks in each frame. They represent the color of solu- 
tions of hemoglobin with percentages from 10 to 120. To obtain interme- 
diate values, colored riders are used, each representing 2.5 and 5 per cent, 
of hemoglobin, when used with the disks from 70 to 120, but twice these values 
if used with the disks from 10 to 60. 



424 



DIAGNOSTIC METHODS. 



The blood is taken in a capillary pipet similar to that of von Fleischl, 
holding 5 cmm. of blood. The diluting chamber has a white background 
of plaster of Paris and, when filled with distilled water in which one pipetful 
of blood has been dissolved, yields a solution of i per cent. When filled the 
cell is covered with a blue glass cover. 

Method. 

The pipet is filled with blood drawn by capillarity from a puncture of 
the ear or finger. Wash the blood from the pipet into the diluting chamber 
by means of water from a medicine dropper, fill the chamber with water, stir well 
with the handle of the pipet, and adjust the cover-glass. A small bubble 

of air should be included as an evidence that 
the chamber was not overfilled. 

Compare the color of this solution with 
that of the standard disks, using the light of a 
candle placed 3 or 4 inches away in such a 
manner that the light strikes the two chambers 
alike. It is well to use the camera tube to 
shield the eyes and enable one to compare 
the tints more accurately. If the color 
matches that of any of the disks, the percent- 
age is read directly from the case. If, how- 
ever, the color is intermediate between two 
disks, the riders must be superimposed on the 
disk of lower percentage, and a second com- 
parison made by using a corresponding plate 
of unstained glass as a rider on the diluting 
chamber. The adjustment of tints and riders 
must be continued until the colors match ex- 
actly. Naturally the variation will equal 2 
per cent, as the ordinary riders are equivalent 
to 2 1/2 per cent, of hemoglobin. It is not necessary for this determination 
that the room be absolutely dark. 

This method is accurate within the limit mentioned (2.5 to 5 per cent.), 
but it is trying, time consuming, and does not equal in accuracy the methods 
of Dare and of Sahli. The instrument is expensive and the disks are prone 
to deteriorate. 

Tallqvist's Hemoglobinometer. 

Tallqvist has introduced a method for the estimation of hemoglobin 
which is so simple that certain writers have been led to recommend it above 
other methods. The principle of this method is essentially the same as Oliver's 
method, although the application is entirely different. Tallqvist compares 
the color of the undiluted blood with that of a series of lithographed standard 
tints, which range by differences of io° from 10 to 100 per cent. These stand- 
ard tints were prepared by matching in water colors the tint of the blood of 
various patients (whose hemoglobin values had been determined with the 




Fig. 130. — Tallqvist's hemoglo- 
binometer. 



THE BLOOD. 425 

von Fleischl instrument) when soaked into standard filter-paper. These tints 
were then lithographed and bound up with sheets of filter-paper, the com- 
bination making a very simple and convenient book which may be easily car- 
ried in the pocket. 

Method. 

Allow a fairly good-sized drop of blood to soak into a portion of the filter- 
paper by holding the edge of the sheet against the drop. Care should be taken 
to allow this to take place very slowly so that the color may be uniform through- 
out. If carefully done it will not be necessary to blot the stain, but some- 
times this is essential. As soon as the stain has lost its humid gloss compare 
its color with that of the lithographed scale. Do not allow the stain to become 
dry, as the color comparisons are disturbed by the coagulation. Hold the 
scale and the stained paper in such a way that the light (daylight only) is well 
reflected from the color scale. The percentage of hemoglobin is then read 
off directly by noticing the point of the scale with which the blood stain ex- 
acdy corresponds. As this scale does not read closer than 10 per cent., the 
intermediate percentages must be judged by difference. Here personal factors 
are of great importance, some workers being so skilled as to detect small 
variations. 

This method can furnish, at best, only an approximate result and has 
nothing in common with the other methods discussed. It is to be advised 
only when some more reliable method is not at hand or when a rough estimate 
only is wanted. It is recommended by some writers as being as generally 
useful and accurate as any of the other methods, but the writer can see no 
advantage whatever in its use, as the results obtainable are, in his opinion, 
not reliable and are not as satisfactory to one who is not especially accustomed 
to such color comparisons. This test would better be limited to rough, pre- 
liminary, approximate determinations than to be used in the more careful 
estimations which clinical work requires. 

In the selection of a method for estimating the hemoglobin of the blood, 
the writer would advise the Fleischl-Miescher instrument for those who are in 
close touch with hospital or clinical laboratory facilities. For the general practi- 
tioner who must make his own determinations under varied circumstances, 
the Dare or the Sahli instrument may be unequivocally recommended, the 
latter having the advantage of cheapness along with accuracy and ease of 
manipulation. 

Variations in the Amount of Hemoglobin. 

The percentage values are misleading, as the hemoglobin varies with 
age and, to some extent, with sex. 1 For this reason it is preferable to obtain 
the absolute amount of hemoglobin, which is done directly by the Miescher 
and, by a very simple calculation, with the Dare or Sahli instruments. In 
those methods which read in direct percentages one may readily calculate 
the absolute values per 100 c.c. of blood by multiplying the percentages obtained 
by 0.1377. I n estimating the true figures for the hemoglobin in the blood of 
1 See Williamson, Jour. Am. Med. Assn., 191 5, LXV, 302. 



426 DIAGNOSTIC METHODS. 

women, it is necessary to add one-eighth to the percentage values as read, 
as female blood runs from 12 1/2 to 20 per cent, less in hemoglobin than does 
the blood of man; likewise for a child we should add one-seventh to the per- 
centage. In this way we correct the readings of the instrument which is 
calibrated against adult male blood. It has been found by comparative tests, 
that the blood of rural residents reaches the normal standards much more 
frequently than does that of their city brothers, this being due, no doubt, to 
the purer fresh air which the country dweller enjoys. 

A relative diminution in the amount of hemoglobin, as determined by 
the above methods, is known as oligochromemia or as achroiocythemia. This 
condition is usually associated with a decrease in the number of erythrocytes 
(oligocythemia), but in cases of chlorosis the diminution of hemoglobin is an 
absolute reduction, each cell showing less hemoglobin than normally and no 
oligocythemia being, as a rule, present. In pernicious anemia, on the other 
hand, each cell may show an absolute increase in hemoglobin, although the 
percentage value is reduced owing to the marked oliogocythemia present 
at the same time. Factors bringing about conditions of hydremia lead to a 
transient oligochromemia, while factors acting as etiologic units in the pro- 
duction of anhydremia lead to a reverse condition of polychromemia. 

Oligochromemia is observed in chlorosis, pernicious anemia, leukemia, 
and secondary anemias, following chronic infections, hemorrhage, malignant 
growths, and some constitutional diseases. It is noted in chronic nephritis, 
chronic enteritis, and mineral poisonings, especially those with lead and 
mercury compounds. It has been stated that low hemoglobin values some- 
times observed in cases which are to have surgical intervention are contra- 
indications to operative treatment as the anesthetics themselves may bring 
on a condition of oligochromemia. While it is wise to watch with extreme 
care cases showing less than 50 per cent, hemoglobin, yet surgical operations 
have been successfully performed on cases with a more marked diminution 
of the hemoglobin. 

Color-index. 

This term is used to indicate the amount of hemoglobin contained in each 
cell, as compared with the amount present when a normal number of erythro- 
cytes obtains. In other words, it is the quotient of the hemoglobin percentage 
divided by the percentage of red cells. This latter factor is obtained by dividing 
the number of red cells, as found by the count, by the number reckoned as 
normal, namely, 5,000,000. A very simple method of getting this percentage 
is to multiply the number of hundreds of thousands of red cells by two; thus 
if 2,650,000 reds were counted we obtain 53 as the percentage of red cells. 
Sahli recommends the use of the term " hemoglobin quotient" or " hemoglobin 
value" for this factor, but the expression color-index has become so general 
that it will be hard to replace. Moreover, the latter term seems to convey 
a more definite idea to most of us than would the substitutes suggested. 

The color-index is normally one; that is, a hemoglobin value of 100 per 
cent, is associated with a blood count of 5,000,000 red cells. We find, how- 



THE BLOOD. 427 

ever, in the different anemias that this figure varies markedly. In those 
anemias, in which the reds are diminished to the same relative extent as is 
the hemoglobin, the index remains normal; while in those cases in which the 
hemoglobin is markedly reduced without a coincident decrease in the reds, 
the value is necessarily less than one. This latter condition is observed es- 
pecially in chlorosis and in splenic anemia, while in the pernicious types of 
anemia we find the diminution of the reds much greater, proportionately, than 
that of the hemoglobin, a high color index consequently obtaining. In such cases 
the index may run from 1.02 to 1.9 as in one case observed by the writer. 

Such variations are the rule, but are not invariable. We may find the 
various anemias showing, at times, very unusual color indices. As this 
factor is intended to show only the relations of the hemoglobin to the cells 
it must not be taken as absolutely diagnostic, but rather as merely significant. 
It must be remembered, moreover, that this figure cannot be absolutely exact 
as it is based on a purely arbitrary number of red cells as the normal value and 
as the instruments for estimating hemoglobin are often improperly standardized. 
The results are, however, comparable and often yield valuable clinical informa- 
tion, if individual, racial, and seasonal variations in both the amount of hemo- 
globin and the number of red cells are taken into consideration. 1 

(C). Proteins of the Blood. 

From the point of view of physiological chemistry a discussion of the various 
protein bodies found in the blood embraces, necessarily, those of the intracellu- 
lar fluid as well as those of the cellular elements. From the clinical stand- 
point, however, the discussion of this field is limited more or less to the proteins 
of the serum. I can, therefore, do little more than refer to the fact that the red 
cells contain, besides the hemoglobin which constitutes about 90 per cent, of their 
organic matter, a nucleoprotein which shows properties resembling those of both 
the globulins and albumins. The proteins of the white cells are still little dif- 
ferentiated. Miescher found five different forms of protein, showing various 
solubility and coagulation relations. Besides these the leucocytes contain, as 
characteristic proteins, nucleins, which are compounds of the phosphoric acid- 
containing nucleinic acid with simple albumins. The metabolism of the nucleins 
is an important factor in various clinical conditions, but I must refer elsewhere 
for such discussions. Little is known regarding the chemical composition of 
the blood-plates. Lowit affirms that they are composed principally of globu- 
lin, while Lilienfeld asserts that their substance belongs to the class of nucleo- 
albumins. 

In the process of coagulation, fibrinogen, a protein of the plasma, is 
converted into fibrin through the influence of thrombin (fibrin ferment), whose 
chemical nature is not absolutely settled; it belongs probably to the class of 
nucleo-proteins, possessing, however, many of the characteristics of the globulins. 
Blood serum, of both physiologic and pathologic types, contains two protein 
bodies, serum globulin and serum albumin. A third body, called by Chabrie 
albumon, has been assumed, but the researches of Drechsel and of Brunner 

1 See Meyer and Butterfield, Arch. Int. Med., 1914, XIV, 94. 



428 DIAGNOSTIC METHODS. 

have shown that this body is not preformed in the serum, but arises from serum 
albumin and serum globulin during the process of coagulation. 

According to Hammarsten, normal human blood serum contains 7.62 per 
cent, of total protein, while Schmidt gives this figure as 8.26 per cent. The 
researches of Reiss, Strauss and Chajes, and more recently of Engel have 
shown that the refraction coefficient of serum, in health, is between 1.3487 
and 1.3517, corresponding to a percentage of 7.74 to 9.13 of protein. The 
introduction of the refraction coefficient into the study of the serum and plasma 
has furnished a method of clinically studying hydremia as well as one by which 
the water content and serum proteins may be easily estimated. 1 These factors 
are of great importance in the study of the various anemias, as we know that 
the serum or plasma is of much more importance, from the pathologic stand- 
point, than are the variations in the cellular structures, which are simply 
manifestations of profound changes in the liquid portions of the blood. 

In this connection we must distinguish between an increase in the proteins 
of the blood (hyperalbuminosis) and a decrease in their amount (hypalbumino- 
sis). The former is observed whenever water is more rapidly withdrawn 
from the system, and so from the blood, than it can be supplied. Such 
conditions are furnished by marked diarrhea, cholera, profuse perspiration, 
and polyuria without extra intake of fluid. This increase in protein content 
is only transient and is a result of mere concentration of the blood, the proteins 
passing out in relatively less amount than does the water. If the above 
conditions are associated with true exudation, then, of course, a hypalbumino- 
sis of transient duration will replace the hyperalbuminosis. This decrease 
in the amount of total proteins is observed whenever direct loss of protein from 
the blood occurs. Thus Becquerel and Rodier observed a diminution of the 
proteins in Bright's disease, cardiac edema, and puerperal fever. Hoppe- 
Seyler noted a loss in melanosarcoma, while Schmidt, von Jaksch, Panum and 
Limbeck, and Pick have reported such a condition in severe anemias and 
leukemias. In most severe infections, as Ewing states, the proteins are but 
slightly reduced. Along with hypalbuminosis we often observe a hydremia 
which may be referable to a direct absorption of fluid from the tissue under 
the influence of a hypertonic state of the blood. 

In contrast to the constancy of the total protein values of the serum, 
we find the relationship between the albumin and globulin markedly disturbed 
at times. These two bodies are in reality not definite chemical compounds, 
but are separable, each into two distinct substances with different solubilities 
and precipitation constants. This fact may have great importance as further 
study is made, inasmuch as Pick has shown that pseudoglobulin has associated 
with it the antitoxins of diphtheria and of tetanus. The normal amount 
of total protein being taken as 7.62 per cent., Hammarsten has shown that 
this percentage is made up of 3.10 per cent, of serum globulin and 4.52 per cent, 
of serum albumin, the relationship of globulin to albumin being as 1:1.5. 

1 See Schorer, Cor.-Bl. f. schweiz. Aerzte., 1913, XLIII, 1523; also, Lowy, Deutsch. Arch, 
f. klin. Med., 1914, CXV, 318; Robertson, Jour. Biol. Chem., 1915, XXII, 233; Tranter and 
Rowe, Jour. Am. Med. Assn., 1915, LXV, 1433. 



THE BLOOD. 429 

This ratio is variable, running, according to Limbeck and Pick, globulin 
16.9 to 38.3 per cent, of total protein, albumin 61.7 to 83.1 per cent, of the 
total albuminous content. Such a wide variation makes it difficult to establish 
any absolute relations in disease. Erben has shown that the albumin remains 
about normal in pernicious anemia, while the globulin is markedly reduced. 
The researches of Estelle, Hoffmann, Halliburton, and Mya and Viglezio show 
marked pathological variations not only in the total protein content, but in the 
so-called "protein quotient," dobSin ' The l atter authors conclude among 
other deductions that the relation of the proteins is greatly changed in disease, 
especially in conditions associated with transudation and exudation, in the 
sense that the globulins 1 are increased while the albumins are diminished. 
In such states globulin is seen to be relatively more resistant and less diffusible 
than is albumin (Gottschalk). That this view is not uniformly applicable 
is noted from the work of Freund who observed in the serum of nephritis a 
relationship of 1 111.3 between the globulin and albumin, instead of the normal 
1:1.5. Ducceschi has reported an interesting observation on this point. He 
found that, during the period preceding the convulsions which follow thyroidec- 
tomy, a percentage increase of albumin as regards globulins obtains, while 
during the convulsions the reverse conditions are present. 

In this discussion I have taken no account of the total protein of the 
whole blood. In its determination we include not only the albumin and 
globulin of the serum, but also the hemoglobin of the red cells, the nucleo- 
proteins of the white cells, and the fibrinogen of the plasma. Traces of albumose 
and peptone (?) have been found in pathological conditions, the former possibly 
in normal states, while still other nitrogenous constituents are commonly 
determined with the proteins. 2 The total protein of the blood has been given 
by von Jaksch as 22.62 per cent., while Limbeck states a percentage of 25. 
So much depends upon the method adopted for its estimation and so much on 
the physiological state of the blood that comparative figures are difficultly ob- 
tainable from the literature. Regarding the determination of the total protein 
and of the globulin and albumin, the reader is referred to the section on Urine. 

Regarding the question of the presence of peptone in the blood, as reported 
by von Jaksch, Freund and Obermayer, and Ludwig, much depends on the 
proper differentiation of the substance found. So much has been called 
peptone that is, in reality, albumose, -that we are uncertain whether peptone 
was present or, if it were, whether it was not formed in the processes used 
or was not a postmortem product. Devoto and Wagner could not confirm 
the finding of true peptone when blood from the living subject was 
examined. It is rather strange that peptone, which reduces the coagulability 
of blood when added in small amounts, should not exert this power in the 
vessels during life were it really present. The fact, however, that peptone 
and albumose have both been repeatedly found in the urine in various condi- 
tions by competent observers points to the probability of the existence of 

1 See Epstein, Jour. Exper. Med., 19 12, XVI, 719. 

2 See Folin and Denis for a discussion of protein metabolism as shown by blood examina- 
tions (Jour. Biol. Chem., 1912, XI, 87 and 161; Ibid., 1912, XII, 141; Ibid., 1913, XIV, 29). 



43° DIAGNOSTIC METHODS. 

these products in the blood, although they may be combined in such a way 
as not to be capable of easy detection. Further elaboration of our methods 
of detection and differentiation may clear up a much disputed field. Bywaters 1 
believes the so-called albumose of the blood to be identical with sero-mucoid. 

(D). Other Nitrogenous Constituents. 
Other nitrogenous bodies than those already discussed are found in the 
blood in normal and, in varying amounts, in abnormal conditions. Owing to 
the introduction of more exact methods of study, especially of the micro-chemical 
methods of Folin and his coworkers, extensive investigation of the non-protein 
nitrogenous elements of the blood has been undertaken, to the end that variations 
in the amount of total non-protein nitrogen as well as of the different specific 
nitrogenous bodies are becoming of great importance in both diagnosis and 
prognosis. It is unquestionably true that a study of the relationship between 
the urinary nitrogen factors and those of the blood gives a much more certain 
index of functional perversion as well as of the extent of this abnormality than 
does the investigation of either group of factors alone. 

Total Nitrogen. 

In the determination of the total nitrogen of the blood, one includes the 
nitrogen referable to the various protein elements as well as that derived from 
the different non-protein nitrogenous bodies. The method adopted in this in- 
vestigation is the usual Kjeldahl method discussed on page 217, using 5 c.c. of 
blood to which a little potassium oxalate is added as withdrawn in order to 
prevent coagulation and to insure exact measurement of the volume desired. 

This factor, in itself, has very little importance. Von Jaksch gives the total 
nitrogen of normal blood serum as 1.37 per cent., while that of the whole blood 
he finds is 3.62 per cent. Variations in these figures are no doubt frequent and 
depend upon changes in the amount of the many different elements making up 
the total blood nitrogen. 

Total Non-protein Nitrogen. 

While the total nitrogen referable to the protein elements of the blood 
shows a practically constant value, we find the non-protein elements subject to 
considerable variation. Although the amounts of the various non-protein 
nitrogen factors are relatively small as compared with those of the protein 
constituents, yet we find the percentage and absolute relationships between the 
total non-protein nitrogen, on the one hand, and the various elements of which 
it is composed, on the other, showing such distinct variations from the normal in 
certain pathological conditions that diagnostic and prognostic deductions may 
be made with increased certainty. For this reason a close study of these factors 
is of great importance. 

Method of Folin and Denis. 

This method is an adaptation to blood examination of the method of Folin 
and Farmer, originally applied to the estimation of nitrogen in the urine. As 
this method was not discussed in the section on Urine, the variations in the 



THE BLOOD. 43 I 

method will be pointed out so that the student may use it in his urinary work, 
should he so desire. 

Obtaining the Blood. 

To the tip of a perfectly clean, dry and accurate pipet attach the usual 
serological needle by means of a short piece of pure gum tubing. Drop a small 
pinch of powdered potassium oxalate into the upper end of the pipet and allow 
it to run down into the tip, in order to prevent coagulation of the blood when 
withdrawn. Connect the upper end of the pipet with a rubber suction tube, 
which is in turn connected with a short glass mouthpiece. Close to the pipet 
place a pinchcock on the rubber tubing, so that the blood may be retained in the 
pipet and a definite amount allowed to run out when desired. 

Insert the needle in the median basilic vein, as usually done in serological 
work, and by suction withdraw the blood to a little above the zero mark of the 
pipet. Remove the needle from the vein and detach it from the pipet. 

Isolation of Non-protein Nitrogen. 
Before the withdrawal of the blood from the vein, fill a 50 c.c. volumetric 
flask about half full of pure methyl alcohol. As soon as the blood is withdrawn, 
as above indicated, adjust the volume of the blood in the pipet to the zero mark 
by means of careful manipulation of the pinchcock. Allow exactly 5 c.c. of the 
oxalated blood to flow from the pipet into the methyl alcohol. Fill the flask 
up to the 50 c.c. mark with methyl alcohol, stopper tightly, shake vigorously 
and allow the mixture to stand for 2 hours in order to permit of separation of 
the protein. 1 Filter the contents of the flask through a dry filter and add to the 
nitrate two or three drops of a saturated alcoholic solution of zinc chlorid. 
Allow this mixture to stand for a few minutes and refilter through a dry filter. 
The filtrate should be perfectly colorless. 

Determination of Non-protein Nitrogen. 
Ten c.c. of this alcoholic filtrate (corresponding to 1 c.c. of original blood) 
are transferred to a large Jena glass test-tube (20 to 25 mm. by 200 mm.). One 
drop of concentrated sulphuric acid, one drop of kerosene and two small beads 
(to prevent bumping) are added, and the methyl alcohol is driven off by immersing 
the test-tube in a beaker of boiling water for 5 to 10 minutes. When the alcohol 
is removed add 1 c.c. of concentrated sulphuric acid, 1 gram of potassium sul- 
phate and a drop of 5 per cent, copper sulphate solution. 2 Boil over a micro- 
burner for about 6 minutes. Allow to cool about 3 minutes until the digestion 
mixture is beginning to become viscous but not solidified. Add about 6 c.c. of 

1 Folin and Denis admit that the methyl alcohol may not precipitate all the protein-like 
material and, further, that the filtrate may not contain all of the non-protein substances. This 
error is unquestionably small and does not seem to call for any revision of the technic. 
Many other protein precipitants have been used by various workers, the latest advocated 
being that adopted by Greenwald of diluting the 5 c.c. of blood with 45 c.c. of 2.5 per cent, 
aqueous solution of trichloracetic acid. 

2 If the total nitrogen of the urine is to be determined by this method, dilute 5 c.c. of urine 
with water to 50 c.c. in a volumetric flask if the specific gravity is over 1,018, or to 25 c.c. if less 
than 1,018. Mix thoroughly and place 1 c.c. of the diluted urine in the Jena test-tube by means 
of the Ostwald pipet. Let the pipet drain against the side of the test-tube for a few seconds and 
then blow it out clean. 



432 DIAGNOSTIC METHODS. 

water, at first a few drops at a time, then more rapidly so as to prevent the 
mixture from solidifying. To the acid solution is then added 3 c.c. of a saturated 
sodium hydrate solution. 1 From this alkalinized digestion mixture the ammonia 
is removed by aspiration, by means of a rapid air current, into a second large 
Jena glass test-tube containing 1 c.c. of N/10 hydrochloric acid and 2 to 3 c.c. of 
water. 2 

For this aeration process the tubes are connected up as follows: Close 
the first tube with a two-holed rubber stopper, through one hole of which passes 
a long tube, roughly standardized to hold 3 c.c. and into which is drawn by 
suction 3 c.c. of saturated sodium hydrate solution, this solution being held in 
the tube by means of a pinchcock attached to the rubber tubing on the top of 
the tube. This long tube reaches nearly to the bottom of the test-tube and is 
later connected with the tube leading from the flask in which the air is washed 
before it goes into the generating tube. Through the second hole of the stopper 
passes, just through the stopper, the tip of a 5 c.c. pipet or a bulbed tube, the 
upper end of which is bent so as to connect, by means of rubber tubing, with a 
long tube passing through the first hole of a two-holed rubber stopper closing the 
second test-tube and reaching to the bottom of the test-tube. Through the 
second hole of this second stopper passes the outlet tube in case compressed air 
is used. If suction is used, this tube is attached to the pump. Care must be 
taken that all the glass tubes, passing through the two-holed stoppers, should not 
be too large for the holes, as otherwise a perfectly tight closure of the tubes is 
impossible. Non-observance of this point will lead to a marked error in the 
determination. It is necessary during the aeration process to prevent the 
concentrated alkaline solution from the first test-tube splashing up into the tube 
leading to the second test-tube, as some of the alkali might creep up along the 
sides of the tube and be carried over into the acid, thus vitiating the results of 
the test. A simple and effective trap consists of a circular piece of rubber cut 
from a two-holed rubber stopper or rubber matting about 1/4 inch thick. This 
is slipped on to the glass tube which reaches to the bottom of the first test-tube. 
This trap should be small enough to go easily into the test-tube and large enough 
to prevent any splash from striking the opening of the exit tube. One or two 
notches may be cut in the edges to permit the liquid, which does get above the 
trap, to flow back easily. It is, of course, understood that the ammonia possibly 
present in the air used for the aspiration must be removed by passing the air 
through a wash bottle containing dilute sulphuric acid, before the air is forced 
into the generating tube. In order to secure perfect absorption of the ammonia, 
as it passes into the standard acid, a miniature Folin absorption bulb should be 
used (see page 231). Further a blank test, using the reagents alone without the 
addition of other material, must be made in order to determine the amount of 
ammonia present in such reagents and the air. This figure is subtracted from 

1 The most convenient method for adding this alkali is to suck it up into the glass tube 
which goes to the bottom of the test-tube and through which the compressed air is forced. 
By means of a short rubber tube and pinchcock the tube is closed and the fluid allowed to run 
out when desired. 

2 In case of urine, aspirate into a 100 c.c. volumetric flask containing about 20 c.c. of water 
and 2 c.c. of N/10 hydrochloric acid. 



THE BLOOD. 433 

the ammonia determined in the later process. A further precaution, to insure 
accuracy, is to run duplicate determinations, so that one may have a check on 
his work. Instead of using the aeration process of Folin one may, if desired, 
substitute the distillation method of Bock and Benedict, 1 passing the ammonia 
into 1 c.c. of N/10 hydrochloric acid diluted with water. 

As soon as the apparatus is ready, attach the air blast and pass the current 
rather moderately for the first 2 minutes and as rapid as the apparatus can 
stand for 8 minutes. After this aeration is complete, disconnect the appa- 
ratus and dilute the contents of the second test-tube with a small amount of 
water (10 c.c). Add 7 to 8 c.c. of diluted Nessler's solution (diluted 1 to 5 
with water just before using) and transfer this mixture quantitatively to a 25 c.c. 
volumetric flask. If a large amount of ammonia is present, as indicated by a 
very deep color, a correspondingly larger amount of Nessler's reagent should be 
added and the mixture diluted to 50 c.c. or even to 100. 

At the same time that one is preparing the above mixture, dilute in a 
100 c.c. volumetric flask 1 mg. of nitrogen in the form of pure ammonium 
sulphate 2 to about 60 c.c. Add about 5 or 6 c.c. of the diluted Nessler's solu- 
tion 3 and make up to the 100 c.c. mark. Mix the contents of each flask by 
thorough shaking and determine the relative density of colors by means of a 
colorimeter, using preferably diffused daylight intead of artificial light. 

In making the colorimeter readings it is important to adjust the unknown 
to that of the standard, both from above and from below the level of the latter. 
Folin advises the use of the Duboscq instrument, but any reliable colorimeter will 
answer the purpose. As in all quantitative colorimetric comparisons, it is 
necessary that the amount of color produced in the unknown solution should be 
reasonably near that of the standard. As we use 1 mg. of nitrogen in the form 
of ammonium sulphate 4 as the standard, the unknown solution should contain 
somewhere between 0.75 and 1.5 mg. The standard may be set at any desired 
point, but 20 mm. represents the point used by Folin with his Duboscq instru- 
ment. The reading of the standard divided by the reading of the unknown and 
multiplied by the dilution gives the nitrogen in milligrams per 100 c.c. of blood. 

Instead of using the above process of Nesslerization, one may aerate the 
ammonia, especially in cases of urine examination, into 5 or 10 c.c. of N/50 
hydrochloric acid diluted with water, and then titrate the remaining acid with 
N/50 sodium hydrate solution using methyl red as an indicator. In this latter 
process 1 c.c. of either N/50 acid or alkali is equivalent to 0.00028 gram of 
nitrogen. 

1 Jour. Biol. Chem., 1915, XX, 47. 

2 Folin advises the purification of even high-grade ammonium sulphate as follows: De- 
compose the ammonium salt with caustic soda and pass the generated ammonia gas into pure 
sulphuric acid by means of the air current. Precipitate the. salt by addition of alcohol, re- 
dissolve in water and again precipitate with alcohol. Finally dry in a desiccator over sulphuric 
acid. 

3 The Nessler's solution, as modified by Winkler, has the following composition: Mercuric 
iodid, 15 grams, potassium iodid, 10 grams; sodium hydrate, 40 grams; ammonia-free water, 
q. s. ad 500 c.c. 

4 In preparing the standard solution of ammonium sulphate, dissolve 4.714 grams of 
absolutely pure and dry ammonium sulphate in 1 liter of ammonia-free water. This gives a 
solution of which 1 c.c. contains 1 mg. of nitrogen. 

28 



434 DIAGNOSTIC METHODS. 

Amount of Non-protein Nitrogen. 

This non-protein nitrogen of the blood has been given various names in 
times past, among which we find "incoagulable nitrogen;" " extractive nitrogen;" 
"filtrate nitrogen;" " non-dialyzable nitrogen;" "rest," "waste," or "retention 
nitrogen." Many figures have been given by various workers for this factor, 
but these depend, to a large extent, on the method employed for its determination 
as well as upon the state of digestion at the time the blood is drawn for examina- 
tion. It has been clearly demonstrated that the "non-protein nitrogen of the 
blood does rise and sink like a tide with reference to absorption from the 
digestive tract." This " digestive rise " is, of course, not a very great one, but it is 
sufficient to necessitate a slightly variable figure for the normal value of non- 
protein nitrogen of the blood. Folin and Denis consider the normal value to be 
between 22 and 26 mg. per 100 c.c. of blood. During digestion the rise is about 
4 mg., so that we may consider a normal non-protein figure to be below 30 mg. 
per 100 c.c. of blood. 

As the kidney is the great regulator of the composition of the blood, main- 
taining a practically constant level of non-protein nitrogen, it is in disorders of 
this organ, especially, that most is to be expected from a study of the variations 
in the non-protein nitrogen of the blood. In the interpretation of an increase in 
the non-protein nitrogen in nephritis Mosenthal considers that four factors must 
be regarded: (1) retention of nitrogen by an insufficient kidney; (2) inspissation 
of blood due to loss of water; (3) increase of protein catabolism; and (4) the 
chemical combination in which the non-protein nitrogen exists in the blood. In 
cases of high fever, associated with marked toxogenic decomposition of protein, 
it might be rational to suppose that these decomposition products would find 
their way into the blood current and increase the non-protein nitrogen of the 
blood. As a matter of fact we do find in pneumonia a considerable increase, as 
a rule, reaching its maximum toward the crisis but bearing no relation to the 
time at which resolution takes place. In very few of the other fevers, however, 
has a rise of any importance been noted. 

Numerous workers have shown that, in the majority of cases, the non- 
protein nitrogen increases with an increasingly severe nephritis. In many cases 
of nephritis, however, the non-protein nitrogen as well as the urea fall within 
normal limits. It is probably true that, in both chronic interstitial and chronic 
diffuse nephritis, cases without symptoms of uremia show normal or only moder- 
ately elevated values. In cases showing considerable hypertension, the non- 
protein nitrogen may be much increased. In cases tending toward uremia or 
showing actual uremia, the values are markedly increased, reaching in some 
cases as high as 350 mg. per 100 c.c. Amounts of 100 mg. or over per 100 c.c. 
are rarely seen in conditions other than uremia, so that this factor assumes great 
importance in diagnosis. Further, the prognostic value of this examination is 
shown in the fact that patients with high non-protein nitrogen do not, as a rule, 
survive for a very long period. Another valuable point in the study of this factor 
is that it furnishes a guide to the proper diet to be allowed nephritics, as cases 
with high retention require restriction of protein. In chronic passive congestion 
of the kidneys there is little or no retention, while in chronic lead poisoning, 



THE BLOOD. 435 

which is so often associated with granular kidneys, considerable retention may 
be observed, but the figure never reaches that shown in uremic conditions. In 
the eclampsias of pregnancy we seldom find a marked increase in the non- 
protein nitrogen, at least so far as reported cases are to be considered. This 
slight retention, in obscure pregnancy, may serve as a point in differential diag- 
nosis from uremia. 1 

Urea. 

This is, of course, the most important of the single elements which make up 
the total non-protein nitrogen of the blood. Its importance lies in the fact that 
its accumulation in the blood runs parallel to that of the total non-protein nitrogen 
and, hence, is of much diagnostic and prognostic value. 

Method of Folin and Denis. 

Ten c.c. of the alcoholic filtrate, obtained in the first part of the preceding 
method for non-protein nitrogen, are accurately measured into large Jena 
test-tube. A drop of dilute acetic acid and two or three drops of kerosene are 
added. Close the test-tube with a two-hole rubber stopper. Through one of 
the holes passes a glass tube drawn out to a capillary several inches long and 
reaching nearly to the bottom of the test-tube. Through the other hole passes 
a short bent glass tube which is connected with a good water pump. The test 
tube is placed in warm water and the vacuum pump started. All of the alcohol 
is removed, by this process, in 10 to 30 minutes. Remove the rubber stopper, 
break off the capillary tube and leave it there. Now add 2 c.c. of 25 per cent, 
acetic acid, a temperature indicator, 2 a small bead, and 7 grams of dry potassium 
acetate. 3 

1 See Folin and Farmer, Jour. Biol. Chem., 1912, XI, 493; Folin and Denis, Ibid., 87, 161, 
503 and 527; Ibid., 1912, XII, 141 and 253; Folin and Lyman, Ibid., 259; Lowy, Ztschr. f 
physiol. Chem., 1912, LXXLX, 349; Folin and Denis, Jour. Biol. Chem., 1913, XIV, 29; 
Ibid., 1913, XVII, 487 and 493; Michaud, Cor.-Bl. f. Schweiz. Aerzte, 1913, XLIII, 14745 
Farr and Austin, Jour. Exper. Med., 1913, XVIII, 228; Karsner and Denis, Ibid., 1914, XIX, 
259; Folin, Denis and Seymour, Arch. Int. Med., 1914, XIII, 224; Agnew, Ibid., 485; Frothing- 
ham and Smillie, Ibid., 1914, XIV, 541; Tileston and Comfort, Ibid., 620; Mosenthal, Ibid., 
844; Gulick, Jour. Biol. Chem., 1914, XVIII, 541; McLean and Selling, Ibid., 1914, XIX, 31; 
Pribram, Zentralbl. f . inn. Med., 1914, XXXV, 153 ; Hertz, Wien. klin. Wchnschr., 1914, XXVII, 
323; Austin and Miller, Jour. Am. Med. Assn., 1914, LXIII, 944; Farr and Krumbhaar, Ibid., 
2214; Farr and Williams, Am. Jour. Obs., 1914, LXX, 614; Am. Jour. Med. Sc, 1914, CXLVII, 
556; Frothingham, Ibid., 1915, CXLIX, 808; Plass, Am. Jour. Obs., 1915, LXXI, 608; Foster, 
Arch. Int. Med., 1915, XV, 356; Fitz, Ibid., 524; Hopkins and Jonas, Ibid., 964; Schlutz and 
Pettibone, Am. Jour. Dis. Child., 1915, X, 206; Tileston and Comfort, Ibid., 278; Hohlweg, 
Mitt. a. d. Grenzgeb.d. Med. u. Chir., 1915, XXVIII, 459; Med. Klin., i9i5,XI, 331; Bock and 
Benedict, Jour. Biol. Chem., 1915, XX, 47; Myers and Fine, Ibid., 391; Greenwald, Ibid., 
1915, XXI, 61; Harding and Warneford, Ibid., 69; Folin, Ibid., 195; Taylor and Hulton, Ibid., 
1915, XXII, 63; Taylor and Lewis, Ibid., 71; Pepper and Austin, Ibid., 81; Folin and Denis, 
Ibid., 1915, XXII, 321; Woods, Arch. Int. Med., 1915, XVI, 577. 

2 This temperature indicator consists of powdered chlorid-iodid of mercury enclosed in a 
sealed glass bulb not over 1 mm. in diameter. The salt may be prepared by heating intimately 
mixed dry mercuric chlorid and mercuric iodid in molecular proportions at 150 to 160 C. for 
6 to 8 hours. The compound is bright red at ordinary temperatures, turns lemon yellow at 
118 C. and melts at 155 C. to a clear dark red liquid. The point at which this indicator melts 
is the optimum point for the decomposition of urea in this test. 

3 This test is applied to urine as follows: Dilute the urine so that 1 c.c. contains 0.75 to 
1.5 mg. of urea nitrogen. Usually dilutions of 1 in 20 or 1 in 10 are adequate. One c.c. of diluted 
urine is transferred by means of an Ostwald pipet to a large Jena test-tube previously charged 
with 7 grams of dry potassium acetate (free from lumps), 1 c.c. of 50 per cent, acetic acid, a 



436 DIAGNOSTIC METHODS. 

Close the test-tube by means of a single-hole rubber stopper carrying an 
empty "calcium chlorid tube" as a condenser. This tube should not have a 
bulb and should be about 25 cm. by 1.5 cm. in size. Suspend the test-tube by 
means of a clamp and heat over a small flame. The mixture usually boils in 
about 2 minutes and the indicator begins to melt, showing that the optimum 
temperature of 153 to 160 C. has been reached. Continue the boiling in a 
gentle even manner for 10 minutes. Remove the tube from the flame and dilute 
the contents with 5 c.c. of water, adding the water by means of a pipet through 
the calcium chlorid tube so as to rinse the sides of the tube and the bottom of the 
stopper. Add 2 c.c. of saturated sodium hydrate solution and, by means of a 
strong air current, drive the liberated ammonia into a large test-tube containing 
1 c.c. of N/10 hydrochloric acid and 2 or 3 c.c. of water. The time required is 
about 10 minutes. Nesslerize the solution in the test-tube by the addition of 
3 c.c. of the diluted Nessler's reagent and make up the volume to 10 c.c. in a 
volumetric flask. Make the color comparison, as in the case of total non-protein 
nitrogen, against the standard ammonium sulphate solution. The calculation 
is made in the same manner as previously given. 

If one prefers not to use the colorimetric method of estimating the ammonia 
driven off, he may, of course, titrate the remaining acidity in the collection tube 
by means of N/50 sodium hydrate solution, using methyl red as an indicator. 
One c.c. of either N/50 acid or alkali is equivalent to 0.00028 gram nitrogen. 
To convert the nitrogen values into those of urea, multiply the nitrogen found 
by 2.143. 

In this method are determined the nitrogen referable to the urea in the 
specimen as well as that derived from the ammonia present, as such, in the 
specimen. In the case of the blood the preformed ammonia factor is sufficiently 
small to be disregarded, but in the case of the urine allowance must be made for 
the nitrogen referable to the preformed ammonia, this latter figure being sub- 
tracted from the nitrogen obtained in the process to obtain the nitrogen referable 
to the urea in the specimen. 

Urease Method. 

This method is an adaptation of the same method discussed in the section 
on urine. Three c.c. of fresh blood or other body fluid are measured with an 
accurate pipet and run into a 100 c.c. heavy Jena test-tube containing 1 c.c. of 
3 per cent, potassium citrate solution to prevent clotting. 0.5 c.c. of urease 
solution (prepared by the method of Van Slyke and Cullen,see Urine, page 226) 
and two or three drops of caprylic alcohol are added. Allow this mixure to 
stand 10 minutes and then add 5 c.c. of saturated potassium carbonate solution. 
Drive off the generated ammonia by means of an air current into 25 c.c. of 
N/100 hydrochloric acid. Titrate back the excess of acid with N/100 sodium 
hydrate solution, using methyl red as an indicator. Each c.c. of N/ 100 acid 

little powdered zinc to prevent bumping, and a temperature indicator. The remaining part of 
the technic is as outlined for the blood, except that the ammonia generated by alkalinizing the 
digestion mixture is driven by the air current into a 100 c.c. volumetric flask containing about 
35 c.c. of water and 2 c.c. of N/10 hydrochloric acid. The Nesslerizing process is as described 
under non-protein nitrogen. 



THE BLOOD. 437 

neutralized indicates 0.00014 gram nitrogen. As 3 c.c. of blood were used in this 
process, we find that each c.c. of N/100 acid neutralized is equivalent to 0.00467 
gram of nitrogen referable to urea or to 0.01 gram of urea per 100 c.c. of 
blood. As fresh blood contains so little ammonia, as such, it may be disre- 
garded in this process. 

Amount of Urea. 

There is much confusion in the literature regarding the amount of urea in 
the blood, as the methods used have been numerous and the urea has been re- 
ported both in terms of urea and of urea nitrogen. The older methods must be 
regarded as inaccurate, so that figures obtained from them cannot be accepted 
at the present time. To obtain comparative figures for these different factors, 
multiply the urea by 0.467 to get the urea nitrogen, or multiply the urea nitrogen 
by 2.143 to derive the urea. 

Folin and Denis, in their analysis of the blood of sixteen normal individuals, 
report that the urea nitrogen varies only between 11 and 13 mg. (approximately 
24 to 28 mg. of urea) per 100 grams of blood. As these specimens of blood were 
collected in the morning 3 to 6 hours after breakfast, the results are to be con- 
sidered as essentially those from a fasting person. During the digestion of a full 
meal containing meat, these values are probably increased by 2 to 3 mg. or 
more of urea nitrogen. Other workers, using the same method, report higher 
figures for the normal urea nitrogen, the values running in some cases as high as 
20 to 25 mg. in 100 c.c. of blood. These higher figures are perhaps above the 
true normal values, but they represent the usual findings in the everyday run of 
clinical material. The urea nitrogen normally constitutes about 50 per" cent, of 
the total non-protein nitrogen. In some cases it may be a trifle higher, but almost 
never is it found to be lower. During digestion the percentage relationship is, 
of course, increased. 

In a general way it is to be expected that variations in the amount of urea 
nitrogen will parallel those of the total non-protein nitrogen. As urea is excreted 
largely through the kidney, serious disturbance of the renal parenchyma should 
be associated with retention of urea. As a matter of fact, it has been found that 
in severe cases of nephritis, accompanied with symptoms of uremia, retention of 
urea is marked, reaching in some cases as high as 250 mg. urea nitrogen per 
100 c.c. of blood and constituting 80 per cent, or more of total non-protein 
nitrogen. It is evident, therefore, that marked retention of urea is a positive 
indication of almost complete renal insufficiency. A large part of the kidney 
parenchyma may be pathological and yet no retention of urea occur, providing 
the remaining renal tissue is sufficient to take care of the ordinary systemic 
requirements. In other words, cases either of interstitial or diffuse nephritis 
may show little or no retention of urea. Should urea retention become marked 
in such cases, uremia is impending owing to the excessive work put upon the 
remaining functionating renal cells. Chronic passive congestion of the kidney 
almost never shows urea retention. Further, one kidney may be so extensively 
diseased as to be incapable of functionating and yet no retention of urea occur, 
as the vicarious activity of the remaining kidrey prevents any accumulation. 



438 DIAGNOSTIC METHODS. 

For this reason, this determination of blood urea can have no importance in 
the study of unilateral renal diseases. 

A number of French investigators have advocated a comparison of the con- 
centration of urea as excreted in the urine with its concentration in the blood, 
believing that a very close relationship exists. Ambard, as a result of his studies, 
has formulated two general laws with reference to this point as follows: (1) 
When the concentration of urea in the urine is constant, the quantity of urea 
excreted in the urine varies proportionately to the square of the concentration of 
urea in the blood. (2) When the concentration of urea in the blood remains 
constant, the quantity excreted in the urine varies inversely as the square root of 
the concentration in the urine. From these laws, corrected for the weight of 
the individual (70 kilos being the standard) and the average concentration of 
urea in the urine (25 grams per liter), Ambard formulated his " uremic constant" 
or "coefficient" which is as follows: 

Ur 

K (constant) 



>»*£ 



in which Ur = grams of urea per liter of blood; D = grams of urea in 24- hour 
specimen of urine; P — weight of patient in kilos; C = concentration or grams 
of urea per liter of urine. 

Normally this coefficient of Ambard is 0.06 to 0.07, being practically 
unaffected by variations in diet as is the amount of urea in the blood. Where the 
amount of urine excreted is very low, this constant tends toward higher values. 
In cases of severe nephritis a large increase in this coefficient is observed as- 
sociated with a relative increase in the concentration of urea in the blood and a 
decrease in the elimination. It would seem, therefore, that this coefficient of 
Ambard might be of considerable value in the study of cases showing little actual 
retention of urea, as far as the absolute amount of the urea is concerned, but in 
which actual evidence of nephritis is present. In other words, may not a 
renal insufficiency be indicated by this coefficient, when the amount of urea 
nitrogen is practically normal? Although we have been somewhat loath 
to accept the conclusions of Ambard and his coworkers, recent studies, es- 
pecially by McLean and Selling, would indicate that an increased coefficient 
may be found in nephritic cases, which do not show any appreciable re- 
tention of urea. In the severe types of nephritis, which are tending toward 
uremia, this coefficient will be markedly increased, just as are the total non- 
protein nitrogen and urea. 

As the liver is concerned to a considerable extent with the formation of urea, 
we might expect to rind in cases of marked hepatic insufficiency a reduction in 
the amount of urea in the blood, just as we do in the urine. However, it is to 
be remembered that the liver is not wholly responsible for the conversion of 
ammonium salts and amino acids into urea, as Folin, Van Slyke and others have 
shown, so that it must be regarded as possible that a practically normal urea 
finding may obtain in the case of the blood and the urinary output be relatively 
much diminished. Further, even in extensive liver disease, such as cirrhosis 



THE BLOOD. 439 

and carcinoma, sufficient hepatic parenchyma may remain to continue the normal 
function of the liver. Recent work by Farr and Krumbhaar would seem to 
indicate that the urea nitrogen of the blood is actually not diminished in hepatic 
disease in contradistinction to the findings reported by various French workers. 1 

Uric Acid. 

Like many substances formed outside the kidneys but excreted by them 
in fairly large quantities, uric acid occurs in the blood in traces too small to yield 
distinct reactions and quantitative results with the usual tests as applied to 
urine. The introduction of newer methods enables us to study much more 
closely the question of the normal uric acid values as well as the pathological 
variations. 

Method of Folin and Denis. 
Ten to 20 c.c. of blood are withdrawn from the median basilic vein by 
means of an accurately graduated syringe, the barrel of which contains a little 
powdered potassium oxalate to prevent clotting. This oxalated blood is then 
added quantitatively to 5 volumes (50 to 100 c.c. depending on the volume of blood) 
of boiling N/100 acetic acid contained in a casserole. This mixture is heated 
to boiling for a moment. Filter while hot and transfer the coagulated material 
from the filter to the casserole by means of a spatula, being careful not to tear 
the filter-paper. Pour over this coagulum about 200 c.c. of boiling water and 
allow to stand 5 minutes. Refilter the mixture through the original filter, 
allowing, the filtrate to mix with the original filtrate. This filtrate should be 
water clear. 2 Further acidify the combined filtrate and wash water with 5 c.c. 
of 50 per cent, acetic acid and carefully evaporate in a deep dish over a free flame 
to about 3 c.c. volume. Pour this small amount of liquid into an ordinary small 
centrifuge tube and wash the dish carefully with two successive portions (of 

1 With reference to various points raised in this section on urea see Ambard, Compt. rend . 
Soc. de Biol., 1910, LXIX, 411 and 506; Assoc, franc, d'urol. Proc.-Verb., 1912, XV, 518; 
Bull, et mem. Soc. med. des hop. de Paris, 1913, XXXVI, 765; Widal and Javal, Ibid., 1911, 
XXXII, 627; Achard, Le role de l'uree en pathologie, Paris, 1912; Ambard and Weill, Jour, de 
Physiol, et de Path, gen., 1912, XTV, 753; Folin and Denis, Jour. Biol. Chem., i9i2,XI, 87; 
Ibid., 1913, XIV, 29; Ibid., 1913, XV, 493; Rowntree and Fitz, Arch. Int. Med., 1913, XI, 258; 
Farr and Austin, Jour. Exper. Med., 1913, XVIII, 228; Courmont, Boulud, Savy and Blanc- 
Perducet, Bull, et mem. Soc. med. d. hop. de Paris, 1913, XXXV, 259; Morel and Mouriquand, 
Ibid., 266; Chauffard, Ibid., 273; Brodin, Compt. rend. Soc. de Biol., 1913, LXXIV, 26; 
Legueu, Ambard and Chabanier, Arch. urol. Clin, de Necker, 1913, I, 275; Legueu, Jour. 
d'Urol., 1914, V, I; Kholzoff, Russky Vrach, 1914, XIII, 465; Studzinski, Ibid., 678; Widal, 
Weill and Radot, Presse med., 1914, XXII, 409 and 565; Jour. d'Urol., 1914, V, 681; Tschert- 
koff, Dutsch, med. Wchnschr., 1914, XL, 1713; Marshall and Davis, Jour. Biol. Chem., 1914, 
XVIII, 53; McLean and Selling, Ibid., 1914, XIX, 31; Folin, Denis and Seymour, Arch. Int. 
Med., 1914, XIII, 224; Agnew, Ibid., 485; Tileston and Comfort, Ibid., 1914, XTV, 620; Farr 
and Krumbhaar, Jour. Am. Med. Assn.,1914, LXIII, 2214; Underhill, New York Med. Jour., 
1915, CII, 662. 

With reference to methods for determination of urea in blood see Folin and Pettibone, Jour. 
Biol. Chem., 1912, XI, 507; Folin and Denis, Ibid., 527; Marshall, Ibid., 1913, XTV, 283; 
Ibid., 1913, XV, 287 and 495; Van Slyke and Cullen, Ibid., 1914, XIX, 211; Jour. Am. Med. 
Assn., 1914, LXII, 1558; Olivieri, Riv. osped., 1914, IV, 221; Kris teller, Ztschr. f. exper. Path, 
u. Therap., 1914, XVI, 496; Siebeck, Deutsch. Arch. f. klin. Med., 1914, CXVI, 58; 
Neumann, Biochem. Ztschr., 1915, LXIX, 134; Combe and Levi, Rev. med. de la Suisse Rom., 
1915, XXXV, 413- 

2 Benedict (Jour. Biol. Chem., 1915, XX, 629) recommends concentration of this filtrate and 
later reprecipitation with a colloidal iron solution (Merck's "Dialyzed Iron") to insure com- 
plete removal of the protein material. 



440 DIAGNOSTIC METHODS. 

about 2 c.c. each) of o.i per cent, lithium carbonate solution, being careful to 
remove any adherent material by means of a rubber-tipped rod. Add the wash- 
ings to the fluid in the centrifuge tube, which should not contain more than 10 
c.c. at the end of the process. To this fluid add 5 drops of 3 per cent, silver 
lactate solution, 2 drops of magnesia mixture (see page 236) and sufficient amount 
of strong ammonium hydrate (10 to 15 drops) to dissolve the silver chlorid 
formed. Centrifuge the mixture for 2 to 3 minutes, pour off the clear superna- 
tant liquid and add to the residue 4 or 5 drops of fresh saturated hydrogen sulphid 
water 1 and 1 drop of concentrated hydrochloric acid. Stir the mixture thor- 
oughly by means of a rod in order to insure complete decomposition of the pre- 
cipitate and liberation of the uric acid. Wash the rod with a drop or two of 
water, so that no material is adherent to the rod. Place the tube in a beaker of 
boiling water for 5 to 10 minutes to drive off the excess of hydrogen sulphid. 
As hydrogen sulphid produces a blue coloration with the special reagent added 
later in the process, it is important that all of this H 2 S be removed. To secure 
this a drop of 0.5 per cent, lead acetate solution is added to the contents in the 
centrifuge tube as it is taken out of the boiling water. Very little, if any, blacken- 
ing should be produced. If such a coloration is obtained, heat the tube in the 
water for another 5 minutes and test again with the lead solution. When all 
the H 2 S is removed centrifuge the tube for 1 to 2 minutes. Transfer the super- 
natant liquid by decantation as completely as possible to a small beaker. Wash 
the inside of the tube with a stream of water from a wash bottle, care being taken 
to disturb the residue at the bottom of the tube as little as possible. Add the 
wash water (which should not exceed 5 c.c.) to the liquid in the beaker and to 
this acid solution containing the uric acid add 2 c.c. of the uric acid reagent 2 
and 10, 15 or 20 c.c. of saturated sodium carbonate solution, depending on 
whether the blue color obtained requires a final dilution of 25, 50 or 100 c.c. 
In determining this point of dilution of the unknown solution, one compares 
roughly the color obtained at this point with that of the standard solution treated 
with the uric acid reagent and sodium carbonate solution. This standard solu- 
tion should be prepared just prior to the addition of the carbonate to the un- 
known solution. Should the color of the unknown solution, when diluted to 
25 c.c. be too deep for that of the standard solution, dilute to 50 c.c. in another 
volumetric flask, which should be at hand ready for this emergency. The same 
statement applies with reference to the dilution to 100 c.c. if necessary. 

As Folin remarks, "No other detail in the colorimetric determination of 
uric acid has presented such difficulties as has the problem of finding a suitable 
standard." The first standard solution used, and one which gives accurate 
results but which is not at all permanent, is a solution of pure uric acid in lithium 
carbonate solution, prepared as follows: Two hundred and fifty mg. of Kahl- 
baum's pure uric acid are washed into a 250 c.c. volumetric flask by means of 25 
to 50 c.c. of water. Add 25 c.c. of a 0.4 per cent, lithium carbonate solution, 
shaking at intervals for an hour before diluting. Make up to 250 c.c. in the flask 
and allow to stand for a few minutes before using. Each c.c. of this solution 

1 Folin and Bell (unpublished method) substitute sodium thiocyanate for hydrogen sulphid. 

2 This uric acid reagent is made as follows: To 750 c.c. of water add 100 grams of sodium 
tungstate and 80 c.c. of 85 per cent, phosphoric acid. Boil gently for 2 hours, using a reflux 
condenser to prevent undue concentration. Cool the solution and dilute it to 1 liter in a 
volumetric flask. Two c.c. of this solution gives the maximum color obtainable with 1 mg. of 
uric acid. 



THE BLOOD. 44I 

contains 1 mg. uric acid. In using this solution for comparison with the 
unknown solution of uric acid, take 1 c.c. of the standard solution, add 5 to 10 
c.c. of water, 2 c.c. of the uric acid reagent and 20 c.c. of saturated sodium 
carbonate solution. Make up to the mark in a 100 c.c. volumetric flask. This 
solution is not advisable for the general laboratory as it necessitates the prepa- 
ration of a fresh solution almost each time a uric acid determination is to be 
made and, hence, causes a needless loss of material owing to the fact that the 
solution does not keep longer than a few days. 

Folin and Denis have, more recently, advised a permanent solution of uric 
acid, which meets the requirements in a general way but which must itself be 
standardized against the above solution of uric acid in lithium carbonate to ob- 
tain its colorimetric value. This solution is made by combining uric acid with 
formaldehyd as follows: One gram of uric acid is placed in a liter volumetric 
flask and dissolved by the addition of 200 c.c. of a 0..4 per cent, solution of lithium 
carbonate. To the solution are added 40 c.c. of 40 per cent, formaldehyd 
solution and the mixture is shaken and allowed to stand for a few minutes. 
Acidify the clear solution with 20 c.c. of normal acetic acid and dilute the 
mixture to the mark with water. The solution should remain perfectly clear 
and the next day (but not before) should be standardized against a freshly 
prepared lithium carbonate solution of uric acid as given above. The color 
produced by 5 c.c. of this solution corresponds very nearly to that obtained from 
1 mg. of uric acid. The colorimetric reading obtained for this solution when 
compared against 1 mg. of pure uric acid, treated with the uric acid reagent 
and the sodium carbonate solution as above, is used as the standard value corre- 
sponding to 1 mg. of pure uric acid. It is seen, therefore, that this solution does 
not possess the reactivity corresponding to the actual amount of uric acid in it, 
so that its value must be determined against known uric acid solutions. This 
standardized permanent solution gives accurate figures and may be used as 
a routine. 

Benedict and Hitchcock have found a standard solution for use in this 
test which seems to be an accurate and extremely permanent one. It has been 
used by the writer many times and is especially recommended for all comparisons 
in this test. The solution is as follows : Nine grams of pure crystallized disodium 
hydrogen phosphate, together with 1 gram of crystallized sodium dihydrogen 
phosphate are dissolved in 200 to 300 c.c. of hot water and the solution is 
filtered, if it was not perfectly clear. Make up the nitrate to a total of about 
500 c.c. with hot water and pour this clear solution upon exactly 200 mg. of pure 
uric acid suspended in a few c.c. of water in a liter volumetric flask. Agitate 
the mixture for a few moments until the uric acid is completely dissolved and 
cool. Now add from a buret exactly 1.4 c.c. of glacial acetic acid and make up 
the contents of the flask to the liter mark with water. Mix the contents thor- 
oughly and add about 5 c.c. of chloroform to prevent development of bacteria 
or moulds. Five c.c. of this solution contain exactly 1 mg. of uric acid. If there 
is any question of the standardizing of this solution, it may be checked up against 
standard solutions of uric acid in lithium carbonate once every month or so. 

The calculation of the results obtained in the colorimetric determinations 



442 DIAGNOSTIC METHODS. 

is not difficult. Treat the unknown solution and the standard solution with the 
prescribed amounts of uric acid reagent and sodium carbonate solution, diluting 
the standard (i mg. of uric acid) to ioo c.c. and the unknown to 25, 50 to or 100 c.c. 
as stated above. Set the standard solution at 20 mm. and compare the colors. 
In making the calculation use the following formula: 

20F 

— ^ = milligrams of uric acid per 100 c.c. of blood. 

In this formula 20 represents the standard reading, R gives the reading in mm. of 
the unknown solution, V represents the volume to which the unknown is diluted, 
and W represents the volume of blood taken for the determination. 

Amount of Uric Acid. 

There is no question but that uric acid is present in appreciable amounts in 
normal blood, although such recognized investigators as Brugsch and Schitten- 
helm state that such is not the case. This divergence of opinion is due to the 
fact that the older methods were far from adequate. In one of their reports, 
Folin and Denis give the normal uric acid values of human blood as varying 
from 1 to 2.5 mg. per 100 grams of blood. Concerning these figures they state 
that " we are not prepared to say that they represent the full variations." A rise 
in the uric acid values undoubtedly occurs following a meal rich in nucle in- 
containing food, but the extent of this "digestive rise" has not been definitely 
established. Weintraud has reported the presence of 5 mg. of uric acid per 
100 c.c. of blood during digestion. This must point to the fact that the kidneys 
have a limited power of excreting uric acid when a surplus is suddenly poured 
into the blood from the digestive tract. Sooner or later, however, this digestive 
accumulation is adjusted and the blood findings become more nearly the figures 
given above as normal. While Folin and Denis have reported increased amounts 
of uric acid in pathological conditions, especially in nephritis, it is interesting 
to note " that there is apparently no relationship between the amount of uric 
acid and the amount of urea or total non-protein nitrogen in human blood." 
While the urea and total non-protein nitrogen of the blood are inversely propor- 
tional to the general efficiency of the kidney, the significance of the uric acid is 
less clear. According to Folin, we may have four distinct classes of blood with 
reference to uric acid: (1) blood in which both uric acid and non-protein nitrogen 
are present in normal amounts ; (2) blood in which with normal amount of uric 
acid we have greatly increased amount of non-protein nitrogen; (3) blood giving 
abnormally high uric acid values with normal amounts of non-protein nitrogen; 
and (4) blood in which abnormally large amounts of both uric acid and non- 
protein nitrogen are present. 

The condition of increase of uric acid in the blood is known as uricacidemia 
or lithemia. In cases of severe nephritis, in which the eliminatory power of the 
kidney is greatly reduced, a relatively marked increase in the uric acid of the 
blood is observed. This increase in nephritis reaches the highest values for the 
uric acid of the blood, being as high as 10 mg. in some cases, and is associated 
with high non-protein nitrogen and urea values. An increase in the uric acid is, 
also, observed in all conditions associated with increased nuclein decomposition. 



THE BLOOD. 443 

In this group we find leukemia, pneumonia, carcinoma and severe febrile condi- 
tions. Further, an increase is noted in practically all cases of chronic lead 
poisoning, due, perhaps, to the associated renal degeneration. 1 

For some time following the teaching of Garrod, an accumulation of uric 
acid in the blood was held to be pathognomonic of gout. That this idea is 
erroneous is seen from the list of conditions in which an increase is observed. 
In fact these other diseases frequently show a uric acid value much higher than 
is ever reached in gout. The usual increase in gout runs from 3 to 7 mg. 
According to Magnus-Levy, there is no increase of uric acid in the blood preced- 
ing or during the gouty attack, but a marked accumulation follows the seizure. 
Folin's findings would seem to indicate that in gout there is almost invariably 
an abnormally high uric acid value while the total non-protein nitrogen is within 
normal limits. In non-gouty arthritis the blood is not infrequently high in uric 
acid, but most cases have abnormally high non-protein nitrogen. Hence, for 
differential diagnosis in a case of suspected gout, the uric acid value is of no 
importance in itself, but must be taken in connection with a high or low non- 
protein nitrogen or urea. 1 

Ammonia. 

Normally the blood contains ammonium compounds to a very slight ex- 
tent, the limits not having yet been determined. This ammonia arises partly 
from the normal pancreatic digestion and finds its way into the circulation as a 
precursor of urea. Moreover, it is especially absorbed into the blood as a product 
of bacterial putrefactive processes in the large intestine, as Folin and Denis 
have shown. As a further source one finds the metabolic activities so adjusted 
that any excess of acids, introduced into the system from without or produced 
within the system by increased protein decomposition, is neutralized up to a 
certain point by a corresponding increase in the ammonia produced. It is, in 
this case, to be regarded as a stage in the deamidization of the proteins, being 
formed when certain nitrogenous groups are liberated from the amino acids, 
which are linked together to form the protein molecule. We are justified, ther- 
fore, in assuming that a preceding increase of ammonium compounds in the 
blood must obtain, to account for an excretion of such an excess of ammonia as 
we find in the urine under certain conditions already discussed. However, this 
point has not been actually demonstrated by experimentation. It is probably 
true that the actual amount of ammonium compounds in the systemic blood is 
almost infinitesimal under normal conditions, although the portal blood is rich 
in such elements. It is evident, therefore, that what ammonia does find its way 

1 See Folin and Macallum, Jour. Biol. Chem., 1912, XIII, 363; Folin and Denis, Ibid., 
469; Ibid., 1913, XIV, 29 and 95; Brugsch and Kristeller, Deutsch. med. Wchnschr., 1914, XL, 
746; Weiss, New York Med. Jour., 1914, C, 180; Adler and Ragle, Boston Med. and Surg. 
Jour., 1914, CLXXI, 769; Kocher, Deutsch. Arch. f. klin. Med., 1914, CXV, 380; Steinitz, 
Deutsch. med. Wchnschr., 1914, XL, 953; Fine and Chace, Jour. Pharmacol, and Exper. 
Therap., 1914, VI, 219; Liefmann, Ztschr. f. Kinderhkde., 1915, XII, 227; Benedict and Hitch, 
cock, Jour. Biol. Chem., 1915, XX, 619; Benedict, Ibid., 629 and 633; Folin and Denis, Arch- 
Int. Med., 1915, XVI, 33; Masse and Zondek, Munch, med. Wchnschr., 1915, LXII, 1105; 
Host, Norsk Mag. f. Laegevidensk, 1915, LXXVI, 1112; Denis (Jour. Pharmacol, and Exper. 
Therap., 1915, VII, 255) shows the increased elimination of uric acid following use of sal- 
icylates; Jorgensen, Ugeskr. f. Laeger, 191 5, LXXVII, 1429. 



444 DIAGNOSTIC METHODS. 

into the blood is, to a considerable extent, rapidly removed therefrom, probably 
through the action of the ammonia- destroying function of the liver. Whether 
an hepatic insufficiency may be demonstrated by later researches on the ammonia 
of the blood is a matter of speculation. Rohde, in her excellent studies with the 
vividiffusion apparatus of Abel, has shown that there is no liberation of ammonia 
comparable to that which takes place under aseptic conditions in shed blood 
and, further, that no ammonia is formed from the diffusible constituents of 
the blood. 

The accurate determination of ammonia in the blood is beset with extreme 
difficulties, owing not only to its very small amount but, also, to the fact that 
the blood decomposes spontaneously to a considerable extent, thus leading to 
an accumulation of extraneous ammonia. This decomposition may, of course, 
be offset to a considerable extent by rapidity in the method adopted for the esti- 
mation of the ammonia. As such determinations do not, at present, offer any 
clinical advantages, the writer is content with reference to the recent literature, 
advising the adoption of the method of Folin and Denis. 1 



Creatinin. 

The number of creatinin and creatin determinations in blood recorded in 
the literature is very small. Only within the last few years has the importance 
of a study of the variations in the creatinin content of the blood been recognized. 
Folin and Denis report that they have found no specific creatinin retention, the 
figures in some 200 cases indicating that the human kidneys remove creatinin 
from the blood with remarkable ease. The normal creatinin content of the blood 
appears to lie between 1 and 2 mg. per 100 c.c. of blood, while the creatin value 
runs as high as 5 or 8 mg. It is, no doubt, true that a retention of creatinin 
occurs, practically speaking, only in nephritis. In such cases we find the crea- 
tinin values paralleling those of non-protein nitrogen and urea, high retention 
being observed only in the severe cases which show a tendency toward uremia. 
Myers and Fine report as high creatinin values as 33 mg. per 100 c.c. of blood 
in cases of this type. Myers and Lough believe the estimation of blood 
creatinin to be of considerable prognostic value, figures from 2.5 to 3 mg. being 
suspicious, those from 3 to 5 mg. being regarded as decidedly unfavorable, while 
values over 5 mg. probably indicate an early fatal termination. From the 
distribution of the non-protein nitrogenous compounds in the blood and urine, 
it would appear that of the three constituents usually estimated creatinin is 
normally the most readily, and uric acid the least readily, eliminated, with urea 
standing in an intermediate position. It seems reasonable to conclude that 
when a noticeable retention of creatinin has occurred, the functional condition 
of the kidney has been markedly impaired. 

As this determination has not, as yet, become a matter of general routine to 
the same extent as have the estimation of non-protein nitrogen and urea, the 

1 Folin and Denis, Jour. Biol. Chem., i9i2,XI, 161 and 527; Taylor and Ringer, Ibid., 1913, 
XIV, 407; Jacobson, Ibid., 1914, XVIII, 133; Fiske and Karsner, Ibid., 381; Rohde, Ibid., 
1915, XXI, 325. 



THE BLOOD. 445 

writer will not discuss the methods to be followed, referring simply to the litera- 
ture for these points. 1 

Amino Acids. 

Much discussion has, for long years, centered around the occurrence of 
amino acids in the blood. It is well known that these nitrogenous bodies, both 
of the mono- and di-amino types, are formed in the intestinal digestion of pro- 
tein material. As the methods of research were for the most part inadequate 
for the detection of these bodies, it was assumed that the blood did not contain 
them but that they were either resynthesized in passing through the absorptive 
process or were at once converted by the liver into urea. Both of these ideas 
have been shown to be fallacious, thanks to the work of Folin, Van Slyke and 
Meyer, Abderhalden (who was formerly one of the staunchest opponents of the 
presence of these bodies in the blood) , and more recently of Abel, Rowntree and 
Turner. Many of these amino acids have been isolated from the blood and 
identified by very careful chemical examination, so that there is no longer any 
doubt of their occurrence in the blood stream. Whether or not variations in the 
amounts of these bodies, leading to a hyper-amino-acidemia or to a hypo- 
amino-acidemia will prove to be of clinical value remains to be seen. The 
methods available for this study are, as yet, not suitable for general clinical work, 
so that the writer will refrain from discussing them. Ellis, Cullen and Van Slyke 
report the amino-acid nitrogen of the blood as varying within certain definite 
limits (4.5 to 8.5 mg. per 100 c.c.) in different individuals and in the same indi- 
vidual at different times. 2 

(E). Carbohydrates. 
The presence of sugar in the blood, both of normal and of abnormal types, 
is a well-demonstrated fact. While this sugar is probably not in the free 
molecular state and is not dialyzable, it is uncertain in just what combination 
it does exist. 3 The discovery of jecorin (a combination of glucose with lecithin) 
by Drechsel shows us that such combinations occur in the blood; yet it has 
not shown that this is the usual blood sugar. The sugar usually present in 
the combined state is glucose, but maltose is occasionally found in the blood 
of nursing mothers, while levulose or pentose may occur both in the blood 
and urine after intake of large amounts of food containing them. 

1 Folin and Denis, Jour. Biol. Chem., 1912, XII, 141; Ibid., 1914, XVII, 475, 487 and 493; 
Neubauer, Munch, med. Wchnschr., 1914, LXI, 857; Myers and Fine, Jour. Biol. Chem., 
1915, XX, 391; Foster, Arch. Int. Med., 1915, XV, 356; Myers and Lough, Ibid., 1915, XVI, 
536; Woods, Ibid., 577. 

2 In this connection see Folin, Jour. Biol. Chem., 1912, XI, 87; Van Slyke, Ibid., 1911, IX, 
185; Ibid., 1912, XII, 275; Ibid., 1913, XVI, 121 and 125; Van Slyke and Meyer, Ibid., 1912, 
XII, 399; Abderhalden, Ztschr. f. physiol. Chem., 1913, LXXXVIII, 478; Kaplan, New York 
Med. Jour., 1913, XCVEI, 1172; Ibid., 1913, XCVIII, 157; Kaplan and McClelland, Ibid., 
1 012; Abel, Rowntree and Turner, Jour. Pharmacol, and Exper. Therap., 1914, V, 611; Ellis, 
Cullen and Van Slyke, Jour. Am. Med. Assn., 1915, LXIV, 126; Gyorgy and Zunz, Jour. Biol. 
Chem., 1915, XXI, 511. 

3 See Lepine and Boulud, Lyon, Med., 1913, XLV, 997; von Hess and McGuigan (Jour. 
Pharmacol, and Exper. Therap., 1914, VI, 45) believe their experiments indicate that the blood 
sugar is free in the plasma and is dialyzable. 



446 DIAGNOSTIC METHODS. 

Glucose. 

The occurrence of glucose in the blood is termed glycemia. This hexose 
is found normally in quantities of 0.5 to 1 part per 1,000. Under pathological 
conditions this amount may increase to as high as nine parts per 1,000, as 
Hoppe-Seyler reports in a case of diabetes. According to Claude-Bernard, when- 
ever the sugar of the blood reaches three parts per 1,000, diuresis and glycosuria 
occur as evidences of the systemic effort to control the hyperglycemia. This 
figure is probably too high, as the later methods 1 of study have given a lower 
value for the normal sugar content and hence for the point at which excretion 
occurs. Indeed, Pavy believes that traces of free glucose in the blood will be 
immediately excreted. While a hyperglycemia may be produced by purely 
alimentary excesses, the most common condition associated with it is diabetes 
mellitus. Here of course many factors, such as the pancreatic, duodenal, 
hepatic, and myogenic influences come into action as causative or contributory 
agents in such production. 

A hyperglycemia is observed following the removal of the pancreas or 
after perversion of its function, as von Mering and Minkowski have shown. 
Also, Pfluger has been able to demonstrate a marked glycosuria and 
hence an hypothetical hyperglycemia, following removal of the duodenum. 
Both the preceding conditions give rise to diabetes and cannot be discussed 
in this place. Several blood tests advocated as diagnostic of diabetes have 
been advanced, especially by Williamson and Bremer, but these will be dis- 
cussed in a later section. 

No doubt can be raised as to the presence in the blood of reducing 
substances other than glucose. According to Paul Mayer, we must assume 
that both normal and abnormal metabolism are associated with the conjugation 
of various aromatic substances with glycuronic acid. These glycuronates 
easily split up into their constituent elements and show reducing properties. 
Whether sugar, in its normal metabolism, must invariably pass through the 
intermediate stage of glycuronic acid is debatable, yet the theory is a fascinating 
one and fits into many obscure points. 

The estimation of the amount of glucose in the blood is fast assuming a 
prominent place in laboratory diagnosis as it enables one to differentiate a di- 
abetic glycosuria, which is always associated with a hyperglycemia, from a 
"renal-diabetic" glycosuria, which is not accompanied by a hyperglycemia. 
While the sugar-content of the blood is much higher in diabetes than in other 
conditions, yet the variations may be sufficient in other disorders to warrant 
such a study. 2 

1 See Reicher and Stein, Ztschr. f. exper. Path. u. Therap., 1912, X, 532; also, Herzfeld, 
Ztschr. f. physiol. Chem., 1912, LXXYII, 420; Jacobsen, Biochem. Ztschr., 1913, LXYI, 471. 
Strouse, Stein and Wiseley (Bull. Johns Hopkins Hosp., 1915, XXVI, 211) show that the normal 
blood sugar varies between 0.04 and 0.12 per cent, with an average of 0.084 per cent., depending 
on the diet of the individual. See, also, Epstein and Baehr, Jour. Biol. Chem., 1914, XVJLLJL, 
21; Wolf and Gutmann, Ztschr. f. klin. Med., 1914, LXXIX, 394; Hirsch, Biochem. Ztschr., 
1915, LXX, 191; Ztschr. f. physiol. Chem., 1915, XCIII, 355; Ibid., 1915, XCIY, 227; Ross and 
McGuigan, Jour. Biol. Chem., 1915, XXII, 407 and 417; Bass, Am. Jour. Dis. Child., 1915, 
IX, 63. 

2 See Scott, Am. Jour. Physiol., 1914, XXXIV, 271; Bergmark, Jahrb. f. Kinderhkde., 
1914, LXXX, 373; Lichtwitz, Berl. klin. Wchnschr., 1914, LI, 1018; Broekmeyer, Deutsch. 



THE BLOOD. 447 

The newer methods introduced are as accurate and not so cumbersome as 
the older, ones, as only a small portion of blood is used. In these methods the 
whole-blood is used, although the serum itself may be employed under certain 
conditions. It must be remembered that the percentage glucose content of the 
whole blood is slightly lower than that of the serum alone (0.076 per cent, for 
the former and 0.096 for the latter), so that slight variations in the values would 
be of little diagnostic importance. In this discussion I select the method of 
Kowarsky 1 as the most serviceable, although the micro-chemical method of 
Bang 2 and the newer method of Lewis and Benedict are excellent. 

Kowarsky's Method. 
Necessary Reagents. 

Solution 1. 

Copper sulphate C. P., 8.00 grams 

Grape sugar C. P., o. 10 gram 

Distilled water, q. s. ad, 200.00 c.c. 

Solution 2. 

Rochelle salt C. P., 40.00 grams 

Sodium hydrate C. P. (in sticks), 30.00 grams 

Distilled water, q. s. ad, 200.00 c.c. 

Solution 3. 

Ferric sulphate C. P., 10.00 grams 

Sulphuric acid C. P., 40.00 c.c. 

Distilled water, q. s. ad, 200.00 c.c. 

Solution 4. 

Potassium permanganate C. P., 1.00 gram 

Distilled water, q. s. ad, 200.00 c.c. 

This solution 4 is used in a ten-fold dilution, which must be made up fresh 
at each determination. The titer of this dilute solution is determined as follows. 
Weigh out exactly 0.1 gram of ammonium oxalate and dissolve it in 100 c.c. of 
distilled water. To 10 c.c. of this solution add 2 c.c. of concentrated sulphuric 
acid and titrate with the diluted permanganate solution to a permanent rose 
color. Divide the arbitrary constant 8.95 by the number of c.c. of KMN0 4 
used to obtain the number of mg. of copper corresponding to each c.c. of this 
solution. 

med. Wchnschr., 1914, XL, 1562; McLean, Jour. Am. Med. Assn., 1914, LXII, 917; Bing and 
Jakobsen, Deutsch. Arch. f. klin. Med., 1914, CXIII, 571; Menke, Ibid., 1914, CXIV, 209; 
Miiller, Ztschr. f. physiol. Chem., 1914, XCI, 287; Kahler, Wien. klin. Wchnschr., 1914, XXVII, 
417; Tachau, Ztschr. f. klin. Med., 1914, LXXIX,42i; Dresel, Ztschr. f. exper. Path. u. Therap., 
1914, XVI, 365; Morita, Arch, f . exper. Path. u. Pharmakol., 1915, LXXVIII, 188; Lauritzen, 
Therap. d. Gegenw., 1915, LVI, 8 and 94; Hopkins, Am. Jour. Med. Sc, 1915, CXLIX, 254; 
Macleod and Pearce, Am. Jour. Physiol., 1915, XXXVIII, 415. 

1 Deutsch. med. Wchnschr., 1913, XXXIX, 1635. 

2 Biochem Ztschr., 1913, XLLX, 19; Munch, med. Wchnschr., 1913, LX, 2277. See, 
also, Dorner, Ztschr. f. klin. Med., 1914, LXXTX, 287. For other methods see Kraus, Lancet, 
1914, I, 1249; Kaminura, Sei-I-Kwai, 1914, XXXIII, 38; Gardner and MacLean, Biochem. 
Jour., 1914, VIII, 391; Maase and Tachau, Ztschr. f. klin. Med., 1914, LXXI, 1; Fitz, Arch. 
Int. Med., 1914, XIV, 133; Epstein, Jour. Am. Med. Assn., 1914, LXIII, 1667; Schaffer, 
Jour. Biol. Chem., 1914, XIX, 285; Lewis and Benedict, Ibid., 1915, XX, 61; Pearce, Ibid., 
1951, XXII, 525; Kahn, Jour. Am. Med. Assn., 1915, LXIV, 241. 



448 DIAGNOSTIC METHODS. 

Other Reagents. 

a. Liquor ferri oxydati dialysati (Merck). This solution is diluted with 
an equal volume of water before being used in the test. 

b. Pulverized Rochelle salt. 

c. A 2 per cent, solution of sodium fluorid. 

Necessary Apparatus. 

For the exact measurement of the blood, flask-shaped tubes (blown from 
glass-tubing) are used. These must be carefully calibrated with two marks 
corresponding to 0.5 and 1 c.c. Small capillary pipets are used to transfer the 
blood from the puncture to the tubes. 

The removal of albumin is done in centrifuge tubes bearing two marks 
at 5 and 10 c.c. 

For the collection and washing of the oxid of copper use small Erlenmeyer 
flasks of 50 c.c. capacity. These flasks are provided with two-holed rubber 
stoppers, through one hole of which passes an Allihn funnel with asbestos filter 
and through the second passes a bent tube for connection with the vacuum pump. 

For titration a buret graduated in 1/20 c.c. should be used. 

Technic. 

By means of the capillary pipets fill two of the flask-shaped tubes with 2 per 
cent, sodium fluorid up to the mark 0.5. Puncture the lobe of the ear or finger. 
Transfer the blood by the capillary pipets to the half -filled tubes until the 
mark 1 is reached. Transfer this accurately measured blood to a centrifuge 
tube and wash out the small tube with distilled water until the blood is all 
removed. Add distilled water up to the mark 5. Add a pinch of Rochelle 
salt, shake lightly until the salt dissolves. Now add about 4 c.c. of solution a 
diluted with an equal volume of water, close the tube with a cork, shake 
thoroughly and allow to stand for five minutes. Fill the tube to the mark 
10 with the diluted solution a and shake strongly. Centrifuge for five or ten 
minutes. 

While the centrifuging is going on, dilute solution 4 ten times. Measure 
into a small Erlenmeyer flask 2 c.c. of solution 1 with a pipet and 2 c.c. of 
solution 2, the latter with a graduate. The centrifuged specimen should now 
be water-clear and albumin-free. Add 5 c.c. of this centrifuged fluid to the mix- 
ture of solutions 1 and 2 and heat to boiling and continue this boiling (lightly) 
for exactly three minutes. Place the hot flask in cold water and allow to stand 
for five minutes. 

Arrange the filtering apparatus and test it with 10 to 20 c. c. of water. Attach 
the suction and filter the cooled copper solution in such a way that most of the 
oxid remains in the flask after the fluid is gone. Wash this residue four or five 
times with distilled water. Disconnect the suction and transfer the stopper and 
filter to the flask containing the residue of copper oxid. Pour on the filter 3 c.c. 
of solution 3 and agitate lightly until the oxid thereon is dissolved. Attach this 
outfit to the suction and add (twice) 5 c.c. of water. The entire filtrate should 
not be more than 10 to 15 c.c. Disconnect the suction, remove the stopper and 
titrate the solution with the dilute solution 4 up to a permanent red color. 



THE BLOOD. 



449 



Calculation. 

The titer of the permanganate solution is determined as above mentioned. 
Multiply the titer by the number of c.c. of solution 4 used and obtain the mg. of 
copper precipitated. From the following table the amount of sugar correspond- 
ing to the copper is read off. 

Subtract from this amount 1 mg. for the sugar contained in solution 1 and 
obtain the true value referable to the amount of blood used. This amount of 
blood is 0.25 c.c. as only one-half of the original 0.5 c.c. of blood taken was added 
to the copper solution. The percentage is determined, therefore, by multi- 
plying the value by 400. Duplicate determinations must be run. 

By this method normal blood contains 0.05 to 0.1 per cent, of glucose. 
Variable values are found in different conditions, in diabetes reaching as high 
as 0.9 per cent. 1 





KOWARSKY'S TABLE OF VALUES IN 


MILLIGRAMS 




Copper. 


Sugar. 


Copper. Sugar. | Copper. ' 


Sugar. 


Copper. 


Sugar. 


i-95 


1.0 


3.30 


i-7 ! 4.65 


2.4 


5.95 


3-1 


2. IS 


1.1 


3-5o 


1.8 4.80 


2.5 


6.15 


3-2 


2-35 


1 .2 


3.7o 


1.9 


5.00 


2.6 


6.35 


3-3 


2.55 


1-3 


3.85 


2.0 


5.20 


2-7 


6.55 


3.4 


2.75 


i.4 


4.05 


2.1 


5.4o 


2.8 


6.70 


3-5 


2 .90 


1-5 


4.25 


2 .2 


5.60 


2-9 






3.10 


1.6 


4-45 


2.3 


5-75 


3.o 







Glycogen. 

This polysaccharid undoubtedly appears in the blood singly or in com- 
bination with albuminous bodies. Salomon, Frerichs, Lepine, Ehrlich, and 
Gabritschewsky have reported it, while Caminer was unable to find it. Hup- 
pert obtained it in quantities ranging from 0.114 to 1.56 grams per 100 parts of 
blood. Much depends on the method used to isolate and determine this sub- 
stance, as it is easily lost by careless manipulation. Certain properties of the 
granules found in the blood, both extra- and intracellularly, have led many to be- 
lieve that glycogen is present as a characteristic in many conditions. There 
is much reason, however, to assume that these granules, which stain brownish 
with iodin, are not glycogen, but rather albuminous bodies of indefinite com- 
position. For a discussion of this subject see the treatment of iodophilia. 

(F). Fats and Fatty Acids. 

The presence of free fats (palmitin, stearin, and olein) in the blood has 
been frequently observed both in health and disease. The relative quantities 
of these fats vary in different animals and are subject to wide variation in the 
same animal under influence of diet. The fat content of the blood under normal 
conditions would appear to be about 0.6 per cent., being higher under influence 
of digestion. 

The presence of an excess of free fat in the blood is referred to as lipemia. 

1 See Roily and Oppermann, Biochem. Ztschr., 1913, XLVIII, 50, 187, 200, 259, 268 and 
471; Ibid., 1913, XLIX, 278; Lampe and Strassner, Med. Klin., 1913, IX, 1462; Bing and Jakob- 
sen, Ugesk.f. Laeger, 1913, LXXV, 1627; Borchardt and Bennigson, Munch, med. Wchnschr., 
1913, LX, 2275; Autenrieth and Montigny, Ibid., 1914, LXI, 1671; Wolff, Deutsch. med. 
Wchnschr., 1915, XLI, 6; Carlson and Ginsburg, Am. Jour. Physiol., 1915, XXXVI, 280. 

29 



450 DIAGNOSTIC METHODS. 

The physiologic variations are more notable than are the pathological, being 
observed after ingestion of a meal rich in fats, in breast-fed children, in preg- 
nant women, and in the obese. 1 Pathologically lipemia more or less permanent 
may be observed in various conditions, as in acute and chronic alcoholism, 
diabetes, arteriosclerosis, chronic nephritis, phthisis, carbon monoxide and 
phosphorus poisoning, gout, typhoid fever, fat embolism following injuries 
of the long bones, pneumonia, leukemia, acute infections, cachexia from inani- 
tion or malignant disease, hepatic diseases, and malaria. It has, therefore, 
little differential diagnostic value. 

The extent of the lipemia may vary from the presence of isolated fat 
droplets to the overloading of the blood to such a degree that it becomes 
salmon-colored, turbid, and milky. This fat may be either the normal fat 
which has been transported from different parts of the body or may be that 
abnormal to the body arising from excess of fat in the diet. It is possible that a 
marked decrease in the lipase of the blood may be a factor in the production of 
lipemia. The fat is soluble in ether and stains black with osmic acid and red 
with Sudan-Ill. Regarding the quantitative estimations of fat, I must refer to 
other sections. 

Concerning the presence of fatty acids in the blood little is known. Traces 
of volatile acids are sometimes present, but probably not as normal constit- 
uents. Gaglio, Spiro, Irisawa, and Berlinerblau report sarcolactic acid as 
a normal finding, while Zweifel finds an excess of this acid in the blood and 
in the urine in cases of toxemia of pregnancy. An excess of fatty acids in the 
blood is known as lipacidemia. Von Jaksch found fatty acids in the blood 
in cases of diabetic coma, leukemia, acute yellow atrophy of the liver, while 
Hougounenq reports the presence of /?-oxybutyric acid in the cadaveric blood 
of a diabetic. It is rational to assume that in all those conditions associated 
with acidosis, fatty acids are present in the blood, as these may be detected in 
large amount in the urine in these states. 

In recent times the variations in the amount of cholesterin in the blood have 
assumed considerable importance. It is, beyond doubt, true that cholesterin 
is present in increased amounts both associated with lipemia and not so associ- 
ated. The cholesterinemia of the alimentary type appears to be dependent 
on the intake of cholesterin-containing food. While the figures for the amount 
of cholesterin in the blood vary to a slight extent, a fairly accurate normal stand- 
ard may be taken as 150 mg. per 100 c.c. of blood. 

A hyper-cholesterinemia obtains in pregnancy, just as does a lipemia. The 
cholesterin gradually rises during gestation, reaches its maximum in the last 
months of pregnancy and drops to normal in 8 to 10 days after delivery, irre- 
spective of lactation. In the eclampsias of pregnancy very high values are noted. 
In chronic nephritis an increase is observed, the degree running parallel to the 
severity of the case. In arteriosclerosis an increase of greater or less extent 

1 Hermann and Neumann, Biochem. Ztschr., 1911, XLIII, 47; Bosco, Policlinico, 1914, 
XXI, 314 and 357; Sakai, Biochem. Ztschr., 1914, LXII, 387; Klein and Dinkin, Ztschr. f. 
physiol. Chem., 1914, XCII, 302; Greenwald, Jour. Biol. Chem., 1913, XIV, 369; Ibid., 191^, 
XXI, 29; Am. Jour. Med. So, 1914, CXLVII, 225; Bloor, Jour. Biol. Chem., 1914, XVII, 377; 
Ibid., 1914, XIX, 1; Ibid., 1915, XXII, 133; Imrie, Ibid., 1915, XX, 87. 



THE BLOOD. 45 1 

is practically always observed. In diabetes mellitus and in tuberculosis one 
obtains relatively high values as a rule. Very high figures (up to 900 mg. per 
100 c.c.) are observed in cholelithiasis, a point of some value in the study of 
pathogenesis of gall-stones. 

An interesting fact is observed in the study of the blood of febrile cases, 
especially of typhoid fever, pneumonia, scarlet fever, etc. In such cases it 
is the rule to find a diminution of cholesterin, the higher the fever the less the 
cholesterin content of the blood. As the temperature diminishes, the cholesterin 
gradually rises, returning to about the normal point as a rule. If the fever be 
long continued, the cholesterin rise in the .period of defervescence goes quite 
appreciably above the normal values. 

The methods of examining the blood for cholesterin are hardly sufficiently 
available for clinical purposes, although they are by no means difficult. Colori- 
metric methods are the ones of choice, as the precipitation methods require 
more blood than is usually at hand. For these methods the writer refers to the 
literature. 1 

(G). Acetone. 
The occurrence in the blood of demonstrable amounts of acetone is known 
as acetonemia. Deichmuller and von Jaksch have found a substance giving 
the reaction of acetone in various conditions, especially in fevers, which finding 
has been confirmed by Reale. Whether acetone is a product of normal 
intermediary metabolism and, as such, is found in many physiologic and. 
pathologic states, must be found in the section on Urine. 2 

(H). Biliary Constituents. 

The conditions in which the biliary constituents, especially the pigments 
and acids, are found in the blood is termed cholemia. 3 It is usually stated 
that these elements are not found in normal blood, but Croftan has shown 
that the bile acids are observed in the blood of healthy subjects. This is not 
unexpected as they are completely absorbed from the intestines and are re- 
excreted in the bile (Weintraud). 4 

1 Lifschiitz, Ztschr. f. physiol. Chem., 1907, L, 437; Ibid., 1907; LILT, 140; Ibid., 1908, 
LVIII, 175; Ibid., 1909, LXIII, 223; Biochem. Ztschr., 1913, LII, 206; Ztschr., f. physiol. 
Chem., 1914, XCI, 309; Ibid., 1914, XCII, 383; Ibid., 1914, XCIII, 209; Windaus, Ibid., 1910, 
LXII, 174; Ibid., 1910, LXV, no; Hanes, Bull. Johns Hopkins Hosp., 1912, XXIII, 77; 
Weston, Jour. Med. Research, 1912, XXVI, 47; Weston and Kent, Ibid., 531; Henes, Deutsch. 
Arch. f. klin. Med., 1913, CXI, 122; Jour. Am. Med. Assn., 1914, LXIII, 146; Thaysen, 
Biochem, Ztschr., 1914, LXII, 89 and 115; von Czyhlarz and Fuchs, Ibid., 131; Antonelli, 
Policlinico, 1914, XXI, 341; Farini, Gazz. d. osp., 1914, XXXV, 993; Fischl, Wien. klin. 
Wchnschf., 1914, XXVII, 982; Anitschkow, Med. Klin., 1914, X, 465; Pribram, Zentralbl. 
f. inn. Med., 1914, XXXVI, 325; Quinan, Calif. State Jour. Med., 1914, XII, 118; Weiss, 
New York Med. Jour., 1914, C, 180; Chauffard and Grigaut, Pfesse med., 1914, XXI, 929; 
Milkowitsch, Russk. Vrach, 1914, XXII, 1911; Cannata, Pediatria, 1915, XXIII, 161; 
Huffmann, Zentralbl. f. Gynak., 1915, XXXIX, 33; Henes, New York State Jour. Med., 
1915, XV, 310. 

2 See Marriott, Jour. Biol. Chem., 1914, XVIII, 507; Jour. Am. Med. Assn., 1914, LXIII, 

397- 

3 See Lehndorff, Prager med. Wchnschr., 1912, XXXVII, 495. 

4 Hymans v. d. Bergh and Snapper (Deutsch. Arch. f. klin. Med., 1913, CX, 540) show 
that the serum always contains traces of bilirubin along with lutein. See, also, Whipple and 
Hooper, Jour. Exper. Med., 1913, XVII, 593 and 612. 



452 DIAGNOSTIC METHODS. 

Pathologically, both the acids and the pigments are found in the blood 
in any condition associated with their appearance in the urine. Oftentimes 
they may be found in the blood when no reaction for them is obtainable in the 
urine. While the bile acids exert marked toxic effects, such as hemolysis, 
the biliary pigments show, as Bouchard, de Bruin, Lugli, and Colosanti have 
demonstrated, certain harmful influences. Flint ascribes the toxic effects 
observed in cholemia to cholesterin, but this idea needs confirmation. The 
most usual condition showing cholemia is jaundice. Whether the blood changes 
observed in the various types of jaundice are due, primarily, to the cholemia 
or to a primary hemolysis or to a combination of these effects as a result of 
intoxication is an unsettled question. 1 

(/). Inorganic Constituents. 

The inorganic composition of the blood shows quite a marked variation 
both under physiologic and pathologic influences. This variation applies 
both to the cellular and intracellular constituents of the blood. Changes 
in the molecular concentration as well as changes in the concentration of 
specific inorganic combinations are observed in the blood of the tw r o sexes, as 
may be seen by consulting the table on page 408. 

Regarding the special significance of the different inorganic constituents 
little is known, but chemical analyses of the blood ash in health and disease 
have shown that the pathological variations are more important as regards the 
chlorids, phosphates, and the iron compounds. 2 

Chlorids. 

Physiologically, a certain (about six parts per mille) concentration of 
sodium chlorid is necessary to hold the proteins in solution, as well as to 
maintain the proper osmotic tension of the serum. The larger the proportion 
of plasma, the greater the percentage of chlorids in the blood. This is true 
only within certain limits as the NaCl-content remains practically constant, 
no matter how large an amount is ingested. This constant value is regulated 
by the increased or diminished renal excretion. 3 

In anemias the chlorids of the blood are usually high, yet, according to 
Limbeck, cases are occasionally found in which normal amounts of sodium 
chlorid obtain. In pneumonia, in which a diminished urinary excretion of 
chlorids is observed, the blood does not show an excess of chlorids but may 
even show a decrease owing to the effects of the exudative process. Diminished 
ingestion of food, vomiting, diarrhea, and general exudative processes may be 
associated with a decrease in the chlorids of the blood, but this is merely 
temporary. In cases of marked nephritis, associated with retention of chlorids, 
the blood may show an excess of sodium chlorid, but this condition soon 

1 See Pel. Deutsch. Arch. f. klin. Med., 1912, CVT, 239; also, Brugsch and Retzlaff, Ztschr. 
f. exper. Path. u. Therap., 1912, XI, 508. 

3 See Weissbein and Aufrecht, Internat. Beitr. z. Path. u. Therap. d. Ernahrungsstor., 
1912, IV, 22. 

3 Bromberg (Jour. d'Urol., 1914, IV, 733) shows that the relation of the chlorids of the blood 
to those of the urine is normally 1 to 2 (hemo-renal index) . Variations in this index are, he 
believes, the first indications of renal insufficiency. 



THE BLOOD. 453 

disappears through the influence of the increased edema. This phase of the 
question, originally advanced by Widal and J aval, has been previously discussed. 

Phosphates. 

These compounds exist in the blood as neutral or alkaline salts of sodium, 
potassium, calcium, 1 and magnesium, as well as in organic combinations in 
the red and white cells in the form of lecithin and nuclein. The inorganic 
phosphates are concerned, at least in part, with the solubility of the proteins. 
Just what variations in the amounts of the organic and inorganic phosphorus 
compounds occur in health and disease is unknown. It is certain that a definite 
phosphorus metabolism exists and that this is characterized, to a great extent, by 
variations in the normal relationship between the compounds of the alkali and 
alkali-earth groups. Just what these relations are in physiologic and pathologic 
states experiment must determine. That the system retains compounds of phos- 
phorus more energetically than it does any other mineral constituents is proven. 
As the phosphates of the blood and of the urine come both from the food and 
from the breaking down of the nuclein-containing protein material of the system, 
large variations are possible under the influence of many factors. The subject 
of the metabolism of phosphorus is taken up under The Urine. 

Iron. 

The iron of the blood is found principally in the hemoglobin which contains 
about 0.42 per cent, of Fe. It is also found in traces in the plasma, and, ac- 
cording to Hammarsten, in the nuclein compounds. The attempt to estimate the 
amount of hemoglobin by determinations of the blood iron have proven failures, 
as no definite relations exist between them. All of the blood iron is not in the 
form of colored compounds and, moreover, some of the derivatives of hemo- 
globin are iron-free. Biernacki has shown that the direct quantitative estimation 
of iron yields higher results than could be obtained by computation from the 
percentage of hemoglobin, this finding being confirmed by Jolles and Jellinek. 

According to Schmidt and Becquerel and Rodier, the amount of iron in 
the blood ranges between 0.056 and 0.058 per cent. Female blood shows a some- 
what lower value, just as it does for hemoglobin. For the estimation of the 
iron of the blood, I must refer to other works for the details, as the method is 
too cumbersome for general clinical use. The principle of the method is based 
on the comparison of the colors of a known solution of iron treated with am- 
monium sulphocyanate solution, with that of a solution of blood iron, obtained 
by incinerating blood, fusing the ash with potassium bisulphate, dissolving 
the fused mass in water, and treating it with sulphocyanate solution. The 
instrument used for the colorimetric tests is called Jolles' ferrometer, the 
amount of iron in 1,000 c.c. of blood being obtained by reference to a table 
accompanying the instrument. If it is desired to obtain the actual percentage by 
weight, the calculation must include the determination of the specific gravity of 
the blood. Having this latter factor, we may use the equation G : V : : 100 : X, 
in which G represents the specific gravity and V the percentage by volume 

1 See Linzenmeier, Zentralbl. f. Gynak., 1913, XXXVII, 958; and Morley, Surg., Gynec 
and Obst., 1913, XVII, 276. 



454 DIAGNOSTIC METHODS. 

of iron which is obtained from the table. The hemoglobin value may be 

found by use of the formula of von Jaksch, Hb = — — , in which M equals 

0.42 

the percentage of iron by weight. 

(/). Blood Gases. 

The gases existing in the blood are oxygen, carbon dioxide, and nitrogen, 
the latter having little importance in the body functions, its amount corre- 
sponding to that which would be absorbed by an equal volume of distilled water, 
namely, 1.8 volumes per cent. 

The amount of oxygen and of C0 2 varies widely, depending on the arterial 
or venous character of the blood, upon the velocity of the blood flow, upon the 
temperature, amount of exercise, etc. Oxygen occurs principally in chemical 
combination with hemoglobin, but a small amount, about one-fourth per cent., 
is present in solution in the plasma. 1 About one-tenth of the C0 2 is held in 
solution in the blood, while the remaining nine-tenths is apportioned as follows, 
one-third loosely bound to the alkalies and hemoglobin of the corpuscles, two- 
thirds held in chemical combination with the alkalies and proteins of the plasma. 

The following table made up from the figures of Setschenow, Ludwig 
and Sczelkow, and Hammarsten, will show the relation between these gases. 

Arterial. Venous. 

Oxygen, 21.6% by volume. 6.8% by volume. 

Carbon dioxid, 4°-3% by volume. 48.0% by volume. 

Nitrogen, i-8% by volume. 1.8% by volume. 

(K). Ferments of the Blood. 

The fact has been well established by Jacobi that the various organs 
and tissues of the body contain ferments which are proteolytic as far as the 
corresponding tissues are concerned, but are usually inactive when applied to 
the proteins of other organs. In other words, these ferments are autolytic, 
but not, as a rule, heterolytic. Much experimental work of former and latter 
years has shown that many metabolic processes, associated with the building 
up and breaking down of various tissue elements, are influenced to a great extent 
by the presence of ferments arising from many sources. 

It is not unreasonable to assume that the blood, like other tissues, contains 
various ferments which have to do with general and special metabolism. 
The work of Schonbein on the oxidases of the blood, of Hanriot and, indirectly, 
of Castle and Loevenhart on the lipolytic ferment, of Lepine, Seegen, and 
Spitzer on the glycolytic ferment shows that such properties are resident in the 
blood. The work of Ascoli and Moreschi, Jochmann and Miiller, and of 
Stern and Eppenstein has opened up an entirely new field of work on the 
proteolytic properties of the leucocytes. This proteolytic ferment action is 
both autolytic and heterolytic and is influenced to a great extent by the checking 
action of the antiferments, which have been so well studied by Opie. Further, 

1 See Peabody (Jour. Exper. Med., 1913, XVIII, 7) for a discussion of the oxygen content 
in pneumonia; also, Preti, Riforma Med., 1915, XXXI ; 432, for variations in blood gases in 
uremia. 



THE BLOOD. 455 

it has been shown that differences exist between the proteolytic properties 
of the many varieties of leucocytes and that the ferments are not always 
heterolytic (Mosse). 

It would lead me too far afield to discuss this subject in detail; hence, 
I must be content with reference to the presence of these substances. 1 Much 
benefit will be forthcoming from a further study of such properties of the blood 
and of certain constituents of the blood. It is to be recalled that ferment 
action may be accountable for the influences of the toxophore and other groups 
of Ehrlich's complement, but this phase must be discussed later. 

(7). Enumeration of Red and White Cells. 

This section of hematological technic is, perhaps, the most perfected and 
most frequently employed. The red and white cells are usually counted, as the 
enumeration of the platelets has little practical or scientific value at the present 
time and the latter technic is not sufficiently perfected to admit of conclusions. 

Various instruments have been introduced for the purpose of counting 
the corpuscles in a given volume of blood. Most of them are based on the 
principle that a layer of diluted blood, of a certain depth, covering a certain 
known space, shows a definite number of corpuscles for every drop of blood 
used. The general method of making the count consists in diluting the 
fresh blood in definite proportions with some indifferent fluid and counting, 
under the microscope, the number of cells in a drop of this diluted blood, 
which is contained in a small glass cell on the floor of which is ruled a series 
of micrometer squares of known dimensions. The cubic contents of the 
cell and the degree of dilution of the blood being known, the number of corpuscles 
counted in any given number of squares of the ruled area may be taken as a 
basis for calculating the total number of cells in a cmm. of blood. Strong 
and Seligmann dispense with a special counting chamber and enumerate 
the cells in a definite quantity of blood diluted in exact proportions with a 
diluent stain and mounted as a permanent specimen. Einhorn and Laporte 
use a somewhat similar method and arrive at very good comparative results. 

The normal number of red cells in the adult male is, approximately, 
5,000,000 per cmm. of blood, while in the female it is somewhat lower, namely, 
4,500,000. Marked variations in this figure are observed in pathological 
conditions and will be discussed in a later section along with the treatment 
of the physiological factors which influence the number and appearance of these 
erythrocytes. In the normal adult the number of white cells varies between 
5,000 and 10,000, the average being 7,500. This figure is subject to both 
physiologic and pathologic influences to a greater extent than are the red cells. 

Many instruments, such as those of Hayem, Gowers, Malassez, Thoma- 
Zeiss, Alferow, and Durham, have been introduced, but the most universally 
used and the best adapted for such investigation is, in the writer's opinion, 

1 Smithies (Jour. Am. Med. Assn., 1912, LIX, 539) has called attention to the presence in 
human blood-serum of an agent (probably a ferment) which shows peptid-splitting properties. 
See Caro (Ztschr. f. klin. Med., 1913, LXXVIII, 286), who finds a lipolytic ferment in the 
serum. Also, Neumann, Biochem. Ztschr., 1913, L, 347; Zamorani, Pediatria, 1914, XXIII, 
401; Porter, Munch, med. Wchnschr., 1914 LXI, 1775. 



456 



DIAGNOSTIC METHODS. 



that of Thoma-Zeiss. This combines certain modifications of the mixing 
pipet of Malassez, the counting chamber of Hayem, and the micrometer 
rulings of Gowers. It gives us, therefore, a most complete instrument for such 
work. 

Hemocytometer of Thoma-Zeiss. 

The blood counter, originated by Thoma and constructed by Zeiss, 
is all that could be desired. Leitz, Reichert, and other makers of optical 
goods have introduced similar types of counters, but our experience with 
them has not been so good. So much depends upon the accuracy with which 
pipets are graduated and upon the trueness of the rulings of the counting 
chamber that we recommend the general use of the Zeiss instrument. 

This instrument consists of a pipet for mixing the blood to a certain 
dilution, a counting chamber by means of which a layer of known depth 
and area is obtained, and a special cover-glass for the chamber. 



I r^ 



Fig. 131. — Thoma-Zeiss counting chamber. 

Pipets. 

The original form of the Thoma apparatus included but one pipet 
this being used for the dilution when both red and white cells were to be 
counted. Experience has shown, however, that the dilution given by this 
pipet is, in most cases, too great to permit of accurate counting of both 
types of cell. 1 A modification has, therefore, been introduced to allow of 
greater accuracy by giving a lower dilution and by furnishing a larger number 
of cells in the counting chamber. 

Erythrocytometer. 

This is the original pipet of Thoma. It consists of a graduated capillary 
tube (A) opening into a dilatation (B), at the opposite end of which is a shorter 
glass tube (C), graduated with a line marked 101 and to which is attached a 
rubber tube with an ivory mouth-piece. This pipet is so graduated that the 
capacity of the ampulla (B) is exactly 100 times that of the capillary tube from 
its point to the line marked 1 and 200 times that from the point to the line marked 
0.5. Other lines, both below and above this latter point, are calibrated on the 
tube, each line representing one-tenth of the capacity of the capillary. In some 
of the pipets, especially that of the Miescher hemoglobinometer, two smaller 
marks- each representing one one-hundredth of the length of the tube are 

1 See Ellermann, Deutsch. Arch. f. klin. Med., 1913, CIX, 378. 



THE BLOOD. 



457 



calibrated on each side of the major divisions. By this means the dilution of 
the blood may be definitely known if the tube be not filled exactly to 
the point described. Those pipets with obtuse ends are much to be preferred 
to those with the more acute angles. In the ampulla is a 
small glass bead which is of service in properly mixing the 
blood with the diluting fluid. This pipet may be used in 
counting both the red and white cells and is all that is abso- 
lutely necessary when one uses the Zappert or Turk count- 
ing chamber. In general work, however, it is used only 
in the count of the red cells as the extreme dilution given 
is better adapted to this estimation than it is to the count- 
ing of the white cells. 

Leucocytometer. 

This is a graduated capillary tube similar to the 
erythrocytometer in construction but having a larger lumen, 
which will permit of lower dilutions. It is graduated into 
ten divisions, with the marks 0.5 and 1 representing these 
measures of the total capacity of the capillary. Above 
the ampulla is a graduation, 11, which is so calibrated 
that blood, drawn to the mark 1 and diluted to 11, gives 
a dilution of 10, while if drawn to the point 0.5 the dilution 
is 20. Those forms of this pipet which have the lower 
end tapering to a fine point ■ giving a gradually decreasing 
lumen, are much to be preferred to the older models. As 
the caliber of this instrument is relatively large, the student N ' 
is cautioned against using too great suction in making the 
dilution, and also against placing too large a drop on the 
counting surface. 1 




The Counting Chamber. 



Fig. 132. — Diluting 
pipets: Erythrocytom- 
eter; leucocytometer 
"This consists of a heavy glass slide, A, on which is with Croy's attach- 

cemented a thick glass ring, B, the surface of which is ment * 

highly polished. This ring surrounds a circular table of glass, D, the height 

of which is just 0.1 mm. less than that of the surrounding ring. Between 

this glass table and the inner edge of the ring is a small ditch, C, to catch the 

drop which may run off from the table and to prevent its flowing up between 

the ring and the cover-glass on the other side of the ditch. On the central 

glass table cross at right angles 21 parallel lines, equidistant, and between 

the extremes of which is exactly 1 mm. Hence we have an area of 1 square 

millimeter divided into four hundred small equal squares. Through each 

fifth row of squares is ruled an extra line, which is not a boundary but is 

merely an aid in keeping one's position in the ruled area. Indicated, not 

1 My colleague, Dr. C. C. Croy, has devised a most excellent and useful pipet attach- 
ment (see above cut), which prevents leakage of the contents during transportation. This 
consists of a strong rubber band with two disks of any kind of thin metal folded on the rubber 
in such a way that the ends of the pipet come in contact with the rubber and press against the 
metal disks. See, also, Saxon and Drummond, Jour. Am. Med. Assn., 1915, LXV, 1182. 



458 



DIAGNOSTIC METHODS. 



bounded, by these extra lines, the square millimeter is divided into 16 units 
of 25 small squares each" (Emerson). 

As the ruled surface of one square millimeter is divided into 400 small 
squares, each small square has an area of 1/400 square mm. The height of 




Fig. 133. — Ruled surface of Thoma-Zeiss counting chamber. (Da Costa.) 

the column of blood being 1/10 mm., the cubic contents of each small square 
is 1/4000 cmm. 

This counting chamber of Thoma does not permit of the counting of a 
sufficiently large number of leucocytes, especially when the dilution has been 
made in the same degree as is used for the enumeration of the red cells. In 
order to overcome this difficulty and also to give a larger ruled area in which 




Fig. 134. — Turk's ruling of the counting chamber. 

the leucocytes may be counted, Zappert has modified the original ruling in 
such a way that a counting surface of 9 sq. mm. is afforded. This 
modification has been improved by Ewing and by Turk in such a way that the 
four large corner squares, each of 1 sq. mm., are subdivided into 16 smaller 
squares, each of which is equal in area to the total 25 smallest squares of the 
Thoma chamber. This latter modification is much the best and is used 
exclusively by the writer. Its advantage in counting both red and white 
cells will be appreciated when the student compares it with the older chamber. 



THE BLOOD. 459 

The sixteen central squares are used in counting the erythrocytes, while the 
entire area may be used in the enumeration of the leucocytes. Simon has 
recently introduced a different modification of the Thoma ruling, which is 
extremely simple and should prove very satisfactory. Other forms have 
been introduced by Burker, Gorjajew and Pappenheim and, more recently, 
by Bass. All give excellent results. 

Before use the counting chamber should be well washed with water and 
carefully dried. Precautions should be taken to see that no lint is left on 
the surface of the glass ring and that no alcohol or ether are used in the cleaning 
process, as these substances loosen the cement with which the glass table is 
fastened to the slide. 
The Cover-glass. 

This is made of heavy polished glass with accurately planed surfaces. 
The ordinary cover-glasses should never be employed with the counting- 
chamber as they are often uneven in surface and do not fit tightly to the slide. 
Moreover, these ordinary cover slips are so thin that the capillarity of the drop 
of blood may bend them down to some extent. The cover-glass must be as 
carefully cleaned and dried as is the chamber. 

Diluting Fluids. 

In order that the blood may be properly examined, it must be diluted 
with a solution which will at the same time prevent coagulation and hemolysis 
and will preserve the corpuscles intact. There are numerous formulae for such 
solutions and the choice is largely a matter of experience. Much will depend 
on whether the red and white cells are both to be counted at the same dilution 
or whether two different pipets are used in making the dilutions. Personally 
the writer prefers the use of two pipets using different diluents, but other 
workers use one pipet and one diluent. The red cells may be destroyed 
by certain fluids leaving the white cells intact or the white cells may be colored 
by the same diluent used in counting the reds. 

Hay em's Solution. 

This solution preserves the red-cells permanently and permits the corpuscles 
to settle slowly, thus furnishing an even distribution of the cells. Moreover, 
it will keep almost indefinitely and does not permit of the development of 
yeast spores which so readily multiply in many of the other diluents. The 
writer can recommend this diluent as the most generally useful of the prepara- 
tions advised. It is made up as follows : 

Mercuric chlorid, 0.500 gram. 

Sodium sulphate, 5.000 grams. 

Sodium chlorid, 1.000 gram. 

Distilled water, 200.000 c.c. 

Owing to the presence of mercuric chlorid, this fluid cannot be mixed 

with an aniline coloring substance to stain the leucocytes and is, therefore, 

not applicable to the combined counting of the red and white cells. 1 

1 Jorgensen (Ztschr. f. klin. Med., 1914, LXXX, 21) believes the results with this solution 
are much improved by diminishing the mercuric chlorid content to 0.1 gram (one-half part per 
liter). 



460 DIAGNOSTIC METHODS. 

Toisson's Fluid. 

Sodium chlorid, 1.000 gram. 

Sodium sulphate, 8.000 grams. 

Neutral glycerin, 30.000 c.c. 

Distilled water, 160.000 c.c. 

Methyl violet 5 B., 0.025 gram. 

The addition of methyl violet serves to color the leucocytes and permits 
of their recognition along with the erythrocytes. Occasionally this fluid 
hemolyzes the red cells and thus invalidates the count. Moreover, it easily 
becomes infected with yeast spores which develop profusely in it. For this 
reason it is advisable to filter the fluid before use, each filtration, however, 
weakening it, so that it becomes after a time useless. 

Other diluents, such as the solutions of Pacini, Lowit, Petrone, Acquisto, 
Edington, and Callison have been used, but the more generally applicable ones 
are those mentioned above. Toisson's fluid is particularly useful when the 
count of reds and whites is to be made in the same specimen. The coloring 
of the leucocytes is not necessary for their recognition, but it is a convenience 
to one who is not making blood counts frequently. Hayem's solution is the 
very best diluent at our command for general purposes. 

If it is desired to count the white cells alone, and this is always wise, a 
1 per cent, solution of acetic acid, to which is added gentian violet to bring 
out the white cells a little more clearly, may be used. This solution destroys 
the red cells and thus gives only the white cells in the preparation. For this 
reason the addition of the gentian violet is unnecessary. Yeast cells develop 
in this solution with more or less readiness, hence one should employ only 
freshly made solutions, as these yeast cells resemble, to some extent, mono- 
nuclear leucocytes and may introduce an error into the count. 

Method of Counting the Corpuscles. 

With this process of counting the cells, whether red or white, there are five 
steps to be taken: 

1. Obtaining the blood. 

2. Diluting and mixing the blood. 

3. Filling the counting chamber. 

4. Counting the cells. 

5. Cleaning the apparatus. 

Erythrocytes. 

(1). Obtaining the Blood. 

As previously stated, the blood may be drawn from a puncture of the 
ear or finger. Personally, the writer always uses the ear, unless some valid 
reason exists for not doing so. As soon as a good-sized drop appears, which 
is obtained without pressure or constriction, the tip of the pipet is placed in 
the drop and is supported by a finger of the left hand, which holds the ear in 
position. The blood is drawn by suction to the mark 0.5, in cases in which 
anemia is not suspected, or to the mark 1 in such cases, as a routine the former 



THE BLOOD. 461 

mark being preferable. As the student will find, some practice is needed to 
stop the column of blood exactly at the point desired. If the blood be drawn 
a little too far, the tip of the pipet may be rubbed with the finger or the excess 
may be shaken down by tapping the tip against a towel or, preferably, the blood 
maybe drawn to the next mark and the necessary correction made in the dilution. 
Unless this error can be corrected, the pipet must be cleaned or a second one 
used. In those pipets which have extra marks, indicating 1 / 100 of the length of 
the capillary, we have a measure which may prevent the necessity of using a 
second test, as the correction may be made in the final calculation. In any 
event, if the blood be not accurately measured, we must reject the determination. 
Moreover, the work should be done rapidly to prevent coagulation of the blood. 
Hence, too much time should not be spent in adjusting the column of blood. 1 

(2). Diluting and Mixing the Blood. 

As soon as the column of blood is adjusted at the desired height of the 
capillary, the tip of the pipet is carefully wiped with the ringers to remove 
any adherent blood and is immediately dipped into the diluting fluid in such 
a way that no portion of the blood is lost. Turk recommends closing the end 
of the pipet with the finger and exerting slight suction on the closed tube in 
order to prevent any loss on immersion of the pipet. I have never seen it 
necessary to use this measure, providing ordinary care is employed in applying 
the suction as soon as the pipet touches the diluent. The diluting fluid 
should stand ready in a small dish or bottle and should be carefully examined 
before use to see that no flocculi or spores are present. 

The diluent should be at once drawn into the capillary by suction. The 
fluid rises slowly in the tube, the pipet which is held vertically being rotated 
between the finger and thumb of the left hand as the fluid rises. By this 
rotation, the diluting fluid is mixed with the blood at once and bubbles of air, 
which often cling to the inside of the tube, are avoided. The glass bead in the 
ampulla serves the purpose of thoroughly mixing the blood and diluent. As 
the column approaches the mark 101 on the upper end of the pipet, care 
should be taken that the aspiration is not too strong. While the error, intro- 
duced by drawing the diluted blood beyond this mark, is not as great as it is 
in drawing the undiluted blood to the mark 0.5, yet both should be avoided. 
When this upper mark is reached, withdraw the pipet from the diluent, close 
the tip with the finger, bend the rubber tubing down over the other end, and 
close this with the thumb. Some prefer to remove the rubber tubing at tbis 
juncture, but this is not at all necessary. Shake the pipet vigorously for at 
least one minute to insure a thoroughly uniform mixing of the contents. If for 
any reason the count is not to be made at once or if it be desired to carry the blood 
to the laboratory for examination, the rubber tubing may be removed and 
the ends of the pipet closed by means of a rubber band. Naturally, before 
the mixture can be used it must be again thoroughly shaken. If the examination 
is to be done at once, proceed as follows. Blow three or four drops from 

1 See Maddox (Jour. Am. Med. Assn., 1913, LX, 663) for the description of a new device 
aiding in filling the pipets. 



462 DIAGNOSTIC METHODS. 

the pipet in order to remove the column of fluid which has remained in 
the capillary and has not mixed with the blood. If Toisson's fluid has been 
used as the diluent, it is better, before blowing out the drops, to allow the 
pipet to lie horizontally for 10 to 15 minutes in order to permit of the 
staining of the leucocytes. 

(3). Filling the Counting Chamber. 

As a rule, it is better, unless Toisson's fluid has been the diluent, to fill 
the counting-chamber at once as errors may creep in by allowing the pipet 
to stand, even though the later mixing may be thorough. A small drop, the 
size of which can be learned only by experience, is blown onto the center 
of the ruled area of the counting chamber. This drop should not be so large 
as to run over into the moat, but should be large enough practically to cover 
the glass table. 

Adjust the cover-glass at once. This is a point in the technic which 
requires considerable practice and must be mastered before accurate results 
can be obtained. Emerson's advice on this point is admirable, " grasp the 
cover-glass by two diagonal corners, place a third corner against the slide 
with the edge of the glass ring as a fulcrum, and hold it in that position by a 
finger of the left hand. By now raising the finger the cover is rotated onto 
the drop rapidly and also in such a way that no air-bubble is left." Breathing 
upon the cover-glass before it is adjusted is often servicable in making this 
preparation. If there has been no dust on the slide or cover-glass and they are 
perfectly clean, the student will observe, providing the adjustment has been 
properly made, a beautiful band of colors known as Newton's rings, which are 
due to a phenomenon of interference of light. If these rings do not appear, 
they may often be brought out by firm pressure on the edges of the cover-glass. 
If they are not persistent, after the pressure has been removed, the adjustment 
of the two polished surfaces must be assumed to be imperfect and the preparation 
rejected. 1 It happens at times that these rings are difficult to obtain and some 
workers state that they are not necessary for an accurate count. The writer 
prefers, however, to reject those slides which do not show these diffraction rings 
than to run the risk of including a possible error. These rings may best be 
seen by holding the slide on a level with the eyes in such a way that the light is 
totally reflected from the surface of the cover-glass. After the slide is properly 
adjusted allow it to stand for three or four minutes before proceeding with 
the next step of the determination, in order to insure the proper settling of 
the corpuscles upon the counting table. 

(4). Counting the Cells. 

Before any count of the cells is attempted, the entire surface covered by 
the blood must be examined with the low-power lens to ascertain whether the 
distribution of the cells is uniform throughout. If not, the slide should be 

1 Eustis (Jour. Am. Med. Assn., 1913, LXT, 1984) advises the following: Grasp the cover- 
glass between thumb and index-finger of right hand. Hold the slide firmly on the table with 
the left hand and rapidly slide the cover-glass across the counting chamber, moving the glass 
in a perfectly horizontal manner. See, also, Hartz, New York Med. Jour., 1915, CI, 612. 



THE BLOOD. 463 

rejected, even though the points mentioned previously have obtained. It is 
much better to stop proceedings at this point than to attempt to equalize an 
uneven distribution by a larger counting area. If the specimen proves satis- 
factory the count may then be undertaken. The lenses best suited to this 
purpose are Bausch & Lomb 1/6, Zeiss D, Leitz 6 or 7, and Reichert 1/6. As the 
student becomes accustomed to cell-counting, he may use a lower objective. 
This has the advantage of bringing the entire field of one sq. mm. into 
focus. A mechanical stage is of some convenience in making the count, but 
the fingers answer quite as well after some practice in manipulating the slide. 

The unit of counting surface is a matter of individual preference. Sahli 
recommends a unit of four small squares, Grawitz and Simon use sixteen of 
these squares, while Cabot advises the use of 36. In common with Turk, 
Ewing, Da Costa, Wood, and Emerson, the writer prefers the unit of 25 small 
squares, as this is the unit of the ruling and the calculation is much simpler 
than with the other units. 

In order to simplify the process of counting, some routine method must 
be used. These methods depend on the worker, but the usual procedure in 
the writer's laboratory is as follows: Adjust the slide upon the table of the 
microscope so that the upper left-hand corner of the central ruled area of 16 
large squares of the Turk chamber is brought into the field in such a manner 
that one may count the cells in the small squares from left to right. If the low- 
power objective is used, the complete group of 16 units will lie within the field 
of observation and may be easily examined. With the higher power, which 
the student may be forced to use in his earlier work, the optical field is neces- 
sarily limited. Count the total number of cells lying within the 25 small squares 
of the single unit. In doing this count the upper row of the unit from left 
to right, drop down a row, and count the cells from right to left and so on until 
the cells in the whole unit have been counted. The accompanying cut will 
indicate the method to be followed. In making this count, cells which touch 
the right hand or lower boundaries of the unit are disregarded, while those 
which touch the upper and left-hand line are included in the count of that square. 
After counting the cells in the upper left-hand unit, count those in the remaining 
fifteen units, thus covering a field of 16 units of 25 small squares each, making 
a total of 400 small squares counted. Turk recommends the counting of eight 
units as a minimum, Emerson advises the counting of the four corner units 
in each of two separate preparations, while Da Costa and others count the cells 
in four groups of units from above downward and repeat with four units not 
adjacent. It seems to the writer that the error is less the larger the area covered, 
and he, therefore, advises the beginner to make the total count of the sixteen 
units, although it is not supposed that one will try to cover up defects in technic 
by this larger counting area. A variation of more than 25 cells in the count 
of the varions units should be taken as evidence that the distribution is not 
perfect. In such cases the count would better be rejected on the ground of 
inaccuracy. After the technic is mastered and the worker has discovered 
just exactly wherein his error lies, a count of 8 or 4 units will suffice. It is 
advisable, where accurate scientific results are indispensable, to clean the slide 



464 



DIAGNOSTIC METHODS. 



and make a second count with a fresh drop of blood so that one may have a 
check on his work. For clinical purposes, however, the count of four units 
will ordinarily be sufficient. 

In ordinary counting it is not necessary to differentiate the red cells from 
the leucocytes, as the error thus introduced is small and may be disregarded. 
In cases, however, which show large leucocyte values, this error will be quite 
appreciable and must be overcome by the following method. All of the cells 
observed may be counted as erythrocytes and the reduction made for the 
number of leucocytes as obtained by the special leucocyte count to be described 
later. In the use of Hayem's diluting fluid, the leucocytes are not colored, 
while the erythrocytes retain their normal yellow color. The leucocytes appear, 
by good illumination of the field, of a bluish tone, are somewhat larger than the 
red cells, and are characterized by a sharper border. These facts will enable 




Fig, 135. — Plan of Counting the Cells. {Da Costa.) 
The small squares are examined in the order indicated by the arrow. 

one after some experience to distinguish the white from the red cells. Toisson's 
fluid stains the leucocytes blue, and may, therefore, be used to outline these cells. 
As a rule, it is better to learn to recognize the leucocytes by differences in 
refraction than to rely on the staining qualities of these cells. In some cases 
the methyl violet of the diluent colors some of the red cells so that they cannot 
be easily distinguished from the white ones. 

The number of cells in the blood is invariably reported as the number 
contained in a cubic millimeter of blood. In making the calculation of this 
number it is necessary to know the number of units counted, the number 
of cells in these units, the area of each small square of the unit, and the degree of 
dilution of the blood. Thus, if 16 units, each of 25 squares, have been counted, 
the total number of small squares is 400, each having a cubic area of 1/4000 cmm. 
The total volume of the units counted is, therefore, 1/10 cmm. It is evident 
that the number of cells in 1 cmm. of blood is 10 times that in the area counted 






THE BLOOD. 465 

over if the blood were undiluted. But the count is always made with diluted 

blood and we must, therefore, take this factor into account. With a dilution 

of 100 multiply the number of cells in 1 cmm. by 100, and with a dilution of 

200 multiply by this factor. Thus, if 2,500 cells were counted in the 400 

small squares and the cubic contents of the units gone over was 1/10 cmm. 

then 1 cmm. of diluted blood would contain 25,000 cells. As the blood was 

diluted 200 times, the total number of cells in one cmm. of undiluted blood 

is 5,000,000. A very simple way of remembering this calculation is to multiply 

the number of cells counted in the total area of 400 small squares by 1,000 if 

the dilution was 100, and by 2,000 if the dilution was 200. 

If the total area of 16 units be not counted, the method of calculation is 

the same but the factors are variable. This method goes as follows: Multiply 

the number of cells counted by the degree of dilution and this result by the 

cubic contents of each small square (4,000). Divide this result by the number 

of small squares counted. Thus, to calculate the number of cells in a cmm. 

of blood when 100 small squares (4 units) were counted at a dilution of 200, 

, . ,i ,1 .• <-n 625 x 200 x 4000 

the count being 625 cells, the equation is as follows: — = 

100 

5,000,000. 

Leucocytes. 

In counting the leucocytes much depends on the sort of ruled slide 
at the disposal of the worker. With the old Thoma chamber at least five 
different drops must be examined in order that a sufficiently large number 
of leucocytes may be counted, while with the Turk cell a counting area of 
9 sq. mm. is afforded for each drop. The more leucocytes counted so 
much the less is the error. It is usually sufficient to count the white cells in 
a single drop, using the Turk chamber, but for scientific purposes three or 
even four drops would better be examined. 

If it is desired to count the leucocytes in the same specimen as the red cells 
the procedure is as follows: Prepare the drop of blood exactly as described for 
counting the red cells, using Toisson's fluid as the diluent and the erythrocy- 
tometer as the diluting pipet. After the reds have been counted, enumerate 
the whites in the entire ruled area of this chamber. In this process the leucocytes 
will be found to be stained a faint blue. It is often advisable, in case of low 
leucocyte counts, to repeat this process with a second drop of blood. The 
calculation by this method is very simple. As the entire ruled area of the Turk 
chamber covers a surface of 9 sq. mm., each equal to the central area used in 
counting the red cells, we have the equivalent of 3,600 small squares in the 
ruled surface. Multiply this figure by the number of drops used to obtain the 
total number of small squares covered by the count. Thus, if 54 leucocytes 
were observed in two drops (7,200 small squares) and the dilution was 200, 

o- 1 .1 .• 54 x 2 °° x 4°°o 

then we have the equation, = 6,000 cells. 

7200 
A second method of calculating the number is to consider each sq. mm. 
of the surface of the Turk chamber as a unit. If, then, the number of cells 
30 



466 DIAGNOSTIC METHODS. 

counted in two drops (18 units) be 54, we divide this number by the number 
of units counted, 18, and multiply the result by 10 (the cubic contents of each 

unit) and then by the dilution. Thus, — - = 6,000. 

1 18 

It is usually preferable in counting the leucocytes to use the special leuco- 
cytometer previously described, as this gives a smaller dilution, and consequently 
a larger number of leucocytes to the counting surface. In using this 
pipet, the blood is drawn to the mark 1 and the diluent, 1 per cent, acetic 
acid, added to the mark 11. This diluent destroys the red cells and brings 
out the leucocytes clearly. The dilution of the blood will thus be 10. If a 
large increase in the number of leucocytes is anticipated, it is better to use 
a dilution of 20, drawing the blood to the mark 0.5, as a routine the dilution of 
10 being, however, preferred. 

In counting the white cells with a dilution of 10, using the Turk chamber, 
it is not as a rule necessary to go over the entire counting field, but more accurate 
results will obtain if this be done. A count of at least 250 cells is advisable, 
while one of 1,000 is more to be preferred in scientific work. The method of 
calculation is the same as given above. Thus if 540 cells were counted in the 

9 sq. mm. of the ruled surface, the dilution being 10, we have — 

y= 6,000. If we have not used the total 9 sq. mm., but have used 4 or any 
other number which may be considered sufficient by the worker, the divisor 
in this division will be the number of units counted. 

If only a Thoma chamber be at hand for counting the cells, two methods 
are available. By the first, several drops may be gone over counting the total 
number of cells in the ruled sq. mm. of the cell. In the second method 
we find the cubical contents of each visual field and then count the leucocytes 
in many of such fields. The method of computing these factors is given by 
Stengel 1 to whose work the author refers the reader who may not have 
access to a Zappert or a Turk chamber. 

In making this leucocyte count great care must be exercised to have 
the diluent fresh and free from yeast spores, which so freely develop in such 
mixtures. If this factor be observed, all the cells seen may be counted as 
leucocytes, but occasionally nucleated red cells may be confusing, especially 
if these be present in large numbers. The physiological condition of the 
patient should always be considered in making a report on a leucocyte count, 
as such factors as digestion and exercise influence this count to a great extent. 

The normal error in making a leucocyte count, with a count of 200 and 
more leucocytes, is about 5 per cent., while in the case of a red count it should 
not be over 3 per cent. Careful work with special attention to all the details 
mentioned will often reduce this error to a lower figure. Naturally, the error 
in counting the leucocytes will be much reduced by using the Turk chamber 
and giving the blood a dilution of 10. There are certain errors due to faulty 
construction both of the pipet and of the counting chamber which remain 
constant in the same apparatus. Hence it is wise to procure the very best 
1 Twentieth Century Practice of Medicine, New York, 1896, VII, 271. 






THE BLOOD. 467 

equipment possible and to test the different portions of the pipet t for such 
errors. In this way a very appreciable difference may be obviated. Not all 
of these blood counters show such variations, but some do, and it is, therefore, 
a matter of moment to know your tools. 

(5). Cleaning the Apparatus. 

This is the last step in the technic of making a blood count. While 
perhaps not as important as some of the other steps, yet if not properly carried 
out it will introduce errors which may prove very annoying. It is readily 
seen that an unclean pipet or counting chamber will interfere with the proper 
manipulations as described above. 

After the cover-glass has been removed from the slide, wash out the chamber 
with distilled water and dry thoroughly by means of a clean piece of linen. 
Place the slide in its proper receptacle, so that it may be conveniently found 
when desired. This may seem a small point, but the author has seen too many 




Fig. 136. — Cross-section of Durham's blood pipet. {Da Costa.) 

T, Glass tube; n, rubber nipple; p, lateral perforation in nipple; c, cork in which 

a capillary pipet is fitted 

slides lost by carelessness in putting them away. The cover-glass is rubbed 
clean and dry and put in the case with the slide. 

Wash out the pipet with water until all of the blood is gone. In some 
cases fine clots will be observed sticking to the side of the tube. Under such 
conditions remove the adherent blood by means of a fine wire or, if this is 
not effective, draw a little strong potassium hydrate solution into the capillary 
and allow it to act on the clot. After the tube is apparently clean, wash it 
out with alcohol and ether. Blow a stream of air through the tube by means 
of compressed air or the suction pump. Be certain that the apparatus is 
perfectly dry and clean, and that the glass bead in the ampulla is freely movable 
as the tube is shaken, before putting the pipet in its case. 

Durham's Hemocytometer. 

Recently Durham has introduced a modification of the older instruments 
for blood-counting. This embodies the principles of the various methods, 
but substitutes a self-measuring capillary pipet for the suction pipet of the 
Thoma apparatus, and special mixing vessels for the dilution of the blood. 
This device makes it possible for one inexperienced in blood-counting accu- 
rately to measure the blood and diluting fluid and thus eliminates the error possi- 
ble with the older pipet. The direct count is made in the counting chamber 
of Thoma. This capillary of Durham is more easily cleaned than that of 
Thoma, thus giving an advantage in cases where several blood examinations 
are to be made in a limited time. While this method of diluting the blood has 
the advantages above mentioned, the writer has never been able to convince 



468 



DIAGNOSTIC METHODS. 




himself that the use of this apparatus gives the student any better results thaD 
he obtains with the Thoma instrument. After some experience, and this is 
needed in any method, the dilution of the blood with the Thoma pipet is 
quite as easily made as with the Durham modification, and the results of the 
count give quite as close checks as do those with the newer method. 

Oliver's Hemocytometer. 

This instrument was intended to furnish a more accurate method of count- 
ing the red cells than was given by the older instruments. The method is 
based on a principle entirely different from that of the older instruments and 
does not afford an actual count of the cells. If blood be 
diluted with a fluid, which preserves the corpuscles, in a 
rectangular test vessel composed of longitudinally striated 
glass, each striation of the glass will act as a lens project- 
ing an image of a candle flame viewed through the sus- 
pension of opaque particles of the blood, providing the 
suspension is of a sufficient dilution to permit of the 
almost unobstructed passage of the rays of light. At the 
proper dilution, these images of the candle flame will 
form a bright streak horizontally across the tube. Ex- 
periments have shown that the development of this bright 
line, on dilution of the blood with Hayem's solution, is an 
accurate measure of the percentage of red cells in the 
specimen examined. The dilution of the blood is mad°. in 
a rectangular glass cylinder by means of a capillary pipet, 
which is washed out with Hayem's solution. The cylinder is graduated into 
divisions from 10 to 120, each division representing 50,000 red corpuscles. 

As this instrument has little clinical value, owing to the fact that the 
error is very great in cases in which the blood is diseased, I refer the reader 
to other works for a description of the method in detail. In the study of the 
physiologic variations of the red cells, this method affords very accurate results, 
giving figures which would be lost with the Thoma instrument. It must be 
remembered that, in this method, as in many others the personal equation plays 
a large role and may account for serious error which is not found with the Thoma 
instrument. Emerson has shown that the variations in the results may be 
as much as 2,000,000 cells when this instrument is compared with the Thoma 
in counting the blood in primary anemia. Baumgarten has proven that varia- 
tions in the size of the cell as well as deformities of the cell will introduce a 
serious error into this method. Ellermann and Erlandsen 1 as well as Dunger 2 
have recently introduced new methods, for making accurate counts of the white 
cells, which may prove very advantageous. 

Counting of the Blood-platelets. 

The technic of counting these cellular elements has been imperfect and the 
results variable. The methods advanced are both direct and indirect. The 

1 Deutsch. Arch. f. klin. Med., 1910, XCVIII, 245. 

2 Munch, med. Wchnschr., 1911, LVIII, 1131. 



Fig. 137. — Oliver's 
hemocytometer. 
(Greene.) 



THE BLOOD. 469 

technic of Determann and of Brodie and Russell belong to the latter class, while 
the method of Helber is a direct one. Recently Wright and Kinnicutt 1 have 
introduced a method which is simple, exact and reliable. The technic is as 
follows: The blood is diluted 1:100 by means of the pipet used for counting the 
red cells and the counting is done in the Thoma-Zeiss counting-chamber, using 
all the precautions previously discussed. The specially thin cover-glass of 
Zeiss, with central excavation, is used to render the platelets clearly visible. 
The diluting fluid consists of 2 parts of a 1:300 aqueous solution of "brilliant 
cresyl blue" and 3 parts of a 1:1,400 aqueous solution of potassium cyanid. 
These two solutions should be fairly fresh, kept separate, mixed and filtered 
just before taking the blood. After the counting-chamber is filled, it is left at 
rest for 10 to 15 minutes in order to allow the blood-platelets to settle to the 
bottom of the chamber and be more easily and accurately counted. The 
platelets appear as sharply outlined, round, oval or elongated lilac-colored 
bodies, some of which form a part of small spheres or globules of hyaline sub- 
stance. The red cells are decolorized and appear as "shadows," while the 
nuclei of the leucocytes are stained a dark blue and their protoplasm light blue. 
This method shows a normal platelet count of 225,000 to 350,000 per cu. mm. 
No constant relations seem to obtain between the variations in the number of 
platelets and of the leucocytes. According to Determann, the ratio between 
the red cells and the blood plates is, on the average, 22:1. The pathologic 
variations in the number of these cellular elements will be discussed in detail in 
a later section. 

III. Morphology of the Blood. 

Before any examination which is concerned with the study of the morpho- 
logical characteristics of the blood can be made, it is essential that all of the 
glassware which comes in contact with the blood should be absolutely clean 
and dry. The glass slides, as they come from the shops, are often coated 
with substances which are removable with difficulty. Moreover, these slides 
are not in all cases perfectly level on both surfaces. It will need but one ex- 
perience with an uneven slide to convince the worker that it is a loss of time 
to attempt the use of such slightly convex or concave slides. The cover- 
glasses should be of the very best quality of glass, should be as thin as possible 
(number o), and three-fourths inch square. The seven-eighths inch square 
covers as also the larger rectangular ones are not as desirable for blood work, 
especially in the examination of fresh specimens. 

The slides and covers should be cleaned with soap and water followed 
by water and alcohol. In some cases it is necessary to soak them in concentrated 
hydrochloric acid for some hours and then wash with water, alcohol, and 
ether, or the ordinary acid-alcohol may be used. After being cleaned they 
should be kept either in 95 per cent, alcohol or, preferably, polished with a 
clean linen cloth or a piece of tissue-paper and kept in dust-proof receptacles. 
It is a wise precaution invariably to polish the slides and covers before use, 

1 Jour. Am. Med. Assn., 1911, LVI, 1457. See, also, Fonio, Deutsch. Ztschr. f. Chir., 1912, 
CXVII, 176. 



470 DIAGNOSTIC METHODS. 

as dust particles are prone to collect even under the best conditions. As a 
rule, it is better to use only new cover-glasses and not attempt to clean them 
after use. The slides may, however, be cleaned by boiling with a strong alkali 
solution, washing with hot water acidified with hydrochloric acid, then with 
hot water, alcohol, and ether. 

After the cover-glasses have been polished it is the best practice to handle 
them only with forceps, as moisture is almost certain to collect on them if the 
fingers be used. This is not only better technic, but more rapid work may 
be done with their use. Two kinds of forceps are necessary in such work. 
The first is one for holding the cover firmly, being found as the locking forceps 
of Ehrlich or the cross-point forceps, while the second is the ordinary pinch 
forceps with which the second cover-glass is handled in making smears. 

(i). Examination of Fresh Blood. 

The examination of fresh blood is a very important part of hematological 
work and should be a routine procedure in every case possible. If the blood 
cannot be examined for several hours after being taken, it is wise not to attempt 
the study of a fresh specimen, as so many changes will occur in such slides that 
no certain findings obtain. The information obtainable from such examination 
of fresh blood often supplements that which one may derive from a study of the 
stained specimens, as some peculiarities, such as the ameboid movement of the 
leucocytes or the motility of the malarial parasite, may be studied only in this way. 

Technic. 

Assuming that the slides and cover-glasses are clean and dry, the ear 
is punctured as previously described. Wipe away the first few drops of blood 
and touch the center of a cover-glass, held with the pinch forceps, to the top 
of the next drop, which should be about the size of a small black-headed pin. 
If this drop be too large the layer of blood will be too thick to permit of proper 
examination. Care should be taken that the cover-glass does not touch the 
skin. Drop this cover onto a slide, which has been warmed by rubbing or 
by passing through a flame. If the glassware be clean, the drop will spread 
evenly in a thin circular layer, not quite to the edge of the cover-glass. Under 
no circumstances should pressure be used to thin the layer or to readjust 
the cover after it has settled on the slide, as artefacts may be easily introduced 
in this way. The slides thus prepared are examined first with a low-power 
lens to obtain an idea of the even distribution of the cells over the entire area. 
The detailed study is carried out with the 1/12 oil immersion lens, but it 
should be remembered that a smaller magnification may give a better general 
survey. These preparations will keep long enough for the purposes of exami- 
nation, but if one wishes to preserve the blood fresh and uncoagulated for 
a longer period it is well to enclose the cover-glass with vaselin or paraffin 
or to use the ordinary hanging-drop chamber as suggested by Rosin and 
Bibergeil. It is sometimes desirable, especially in the study of malarial 
parasites, to use a warm stage or a warm chamber. If, however, the specimen 
is examined soon after its preparation, no such precaution is necessary, provided 
the room is not cold. 



PLATE XVII. 




Kathan'** 



Fresh Normal Blood. (Zeiss Ocular 4, Objective DD.) 



THE BLOOD. 47 I 

In order to judge of the changes which abnormal blood may show in the 
fresh state, one must be thoroughly familiar with the appearance of normal 
blood. This latter knowledge can be obtained only by frequent study of 
fresh normal specimens and not from any text-book description. To attempt to 
learn without microscopic study the size, shape, color, and refraction of the red 
and white cells, the relation of the blood-plates to fibrin formation, the number 
of the various cells and their ratio to one another, would be absolute idiocy. 

An examination of the fresh blood as described above gives information 
regarding the presence or absence of the malarial parasite, the spirochete 
of relapsing fever, the filaria, and trypanosomes. It affords evidence of increased 
or decreased rouleaux formation, number, deformities, and degenerations, 
as well as of the amount of hemoglobin of the red cells; the presence of a 
leucocytosis or of a leucopenia and of ameboid movement of the leucocytes. 
However, care must be taken to avoid premature conclusions from such study 
and to institute further examinations of the stained specimen to clear up doubt- 
ful points. ' The observer must be on his guard lest he mistake the normal 
Brownian movement in the protoplasm of the cells for ameboid or parasitic 
movement, Curious phenomena are observed in the fresh specimen as the 
blood dries and should not be misinterpreted. The various characteristics 
of fresh blood will be taken up in detail later. The introduction of the ultra- 
condenser or dark-field illuminator has opened up a field of great possibilities 
in the examination of specimens of fresh blood, especially when malarial 
parasites or spirochaetae pallidse are suspected. 





Fig. 138. — Preparation of smears with two glass slides. {Da Costa.) 

(2). Preparation of Smears. 

To prepare blood smears, which are to be later examined in the stained 
condition, one may spread the blood in capillary layers on slides or between 
cover-glasses. The former method is the one used in the writer's laboratory 
and has given excellent and satisfactory results. A fair-sized drop of blood is 
collected on one end of a clean dry slide, held between the thumb and second 
and third finger of the left hand. A second slide is held in the same manner 
by the right hand, but at an angle of 45 degrees to the first one and touching 
the drop of blood. Allow the blood to spread out by capillarity along the 
edge of the second slide. As soon as this occurs, draw the drop of blood 
along the first slide with a clean sweep, exerting little pressure with the second 
slide and maintaining the angle of 45 between the two slides, allowing the 
second slide to rest rather upon the blood than upon the slide (see cut). In the 
process, as recommended by some writers, the second slide is gradually drawn 



47 2 



DIAGNOSTIC METHODS. 



into a position perpendicular to the first one. This procedure does not yield, 
in the writer's hands, as good results as the former method, as it is more difficult 
to maintain equal pressure, the smear being as a result too thick or too thin 
in places. Instead of a slide, a cigarette paper may be used as a spreader 
and gives good results. This method of making blood smears has the advan- 
tage of offering a large surface for examination, of making smears which are 
fairly uniform after some practice, of dispensing with the necessity of mounting 
the specimen, and of permitting the fixation of the smear in the free flame. 
It is less expensive than the method to be described later and permits of the 




Fig. 139. — Preparation of blood smear with cigarette paper. {Da Costa.) 

cleaning and later use of the slides. The beginner may find, on examining his 
early specimens made by this method, that the leucocytes collect at the distal 
end of the smear and that the general surface contains few white cells. This 
is due to the use of undue pressure in making the smear, and may be avoided 
by proper attention to this detail. 

A second method which has many advocates is the use of two cover- 
glasses. One clean, dry cover-glass, which should not be too large (preferably 
three-fourths inch square or the larger rectangular slips), is held by the Ehrlich 
or cross-bladed forceps or, as some advise, between the thumb and first finger 
of the left hand. The other cover, held in the pinch forceps or between the 
thumb and first finger of the right hand, is touched to the drop of blood as it wells 
from the puncture in the ear. This second cover is then dropped at once upon 




Fig. 140. — Ehrlich forceps 

the first in such a way that the corners of the two glasses do not coincide. If 
the glasses are clean the blood spreads out evenly in a thin capillary layer be- 
tween them. As soon as the spreading is complete, the two covers are drawn 
apart, in a line parallel to the plane of their surfaces, by a steady, quick motion, 
being sure to avoid lifting them apart. This manipulation can be learned 
only by practice and never from any description. If the fingers are dry and 
if care be taken to touch only the corners of the covers the forceps need not 
be used in separating the covers, but it must be remembered that moisture will 
cause changes in the specimen. It is, therefore, advisable to use the forceps 
in this part of the technic unless the rectangular slips be used. As soon 
as the covers are separated, they are allowed to dry in the air or by waving 
them two or three times to and fro. They should be at once placed in a clean, 



THE BLOQD. 



473 



closed receptacle, such as a Petri dish, until ready for the later complete fixation 
and staining, as dust will collect upon them or flies may attack them if left in 
the open air. It is rarely necessary to fix these smears at once, but with some 
stains such treatment is advisable. This second method of making the blood 
smears is more difficult than the first, is not so reliable, does not give as great 
a surface for examination, and always shows the lower cover-glass better and 
more uniformly spread than the upper. 

Smears made by either of these methods should be uniform throughout 
with the exception of the edges, which should never be used as they are too 
thick for allowing definite conclusions to be drawn. The red cells should 
lie on their broad surface, should not be in rouleaux except at the edges, and 
should not show deformities due to errors in technic. The leucocytes 




Fig. 141 — Pinch forceps. 

frequently collect at the edges of the specimen if too great pressure be used 
in making the smear, while the platelets always collect at the point first touched 
by the second slide or cover-glass. The preparation of thin even smears is 
necessary for the proper carrying out of the later technic. Those specimens 
which are irregular or are too thick would better be discarded, as the time 
consumed in studying such specimens will not be compensated for by the 
results obtained. It is much better to make several smears than to be content 
with a few bad ones. A little experience with poor smears will convince the 
worker that it is advisable to use great care in preparing them, especially if 
a differential count is to be made or if a study of the degenerations and deformi- 
ties of the cells is to be undertaken. 1 

(3). Fixation of Smears. 

Before any staining of the cellular elements of the smear takes place, 
the protein constituents of the blood must be coagulated by exposing the 
air-dried film to the action of a high degree of heat or to that of various chemical 
reagents. The selection of the method of fixation will depend to a great extent 
upon the stain to be used later. Fixation is always essential if aqueous stains 
are used, while it is not so necessary if strong alcoholic solutions are employed. 
In the use of the different modifications of the Romanowsky stain, the fixation 
is done by the methyl alcohol employed as a solvent for the various stains. 
As a general rule, fixation by heat is preferable to that by chemicals, as artefacts 
are less prone to appear, providing the degree of heat is carefully regulated. 

Fixation by Heat. 

This method, which is the most difficult to use and which is at the same 
time the best, is the only one which is reliable when Ehrlich's triple stain is to 
1 See Schilling, Deutsch. med. Wchnschr., 1913, XXXIX, 1985. 



474 



DIAGNOSTIC METHODS. 



be employed. The principle is as follows : The air-dried specimen is subjected 
to the action of a temperature of no to 150 , for a more or less varying length 
of time, depending on the experience of the special worker. The lower the 
temperature the longer must its action be exerted. 

The apparatus most frequently employed is the copper plate introduced 
by Ehrlich. This is an unpolished triangular plate of copper about 3 mm. 
thick, 30 to 50 cm. long, and 10 cm. wide, which is held in position by vertical 
standards. It is heated by an alcohol lamp or a Bunsen burner placed under 
the narrower end until the temperature of the plate becomes constant, the 
parts nearer the name being naturally warmer than those more remote. The 
temperature of the different portions of the plate may be readily found by 
determining the points at which water (100), toluol (no), xylol (140), or of 
turpentine (150) boil. The slides or cover- glasses are then placed, smeared 
side up, at the desired point (the outer margin of the glass being three-fourths 
inch from the boiling-point and toward the flame). Just how long a period is 
necessary, at the temperature selected, to give a perfect fixation will depend 

upon the age of the specimen and upon the 
condition to be studied. It is good practice 
to place several slides at the point desired for 
a period of one hour and then remove a slide 
at intervals of 15 minutes thereafter. One of 
the specimens is sure to be good by this method 
and the remaining ones may, therefore, be 
properly heated. Freshly made specimens re- 
quire longer heating than the old ones, while 
normal blood requires a longer exposure than 
abnormal specimens. As a rule, the specimens 
require, when the triple stain is to be used 
(and this is the one most frequently employed 
with heat fixation), from one to one and one- 
half hours at a temperature of no, although 
some workers use only a few minutes' (one to 
three) exposure to such temperatures. The 
higher the temperature the less time is essential. Rubinstein uses a point at 
which a drop of water does not boil, but assumes the spheroidal state (so-called 
Leidenfrost phenomenon) and places the slides, with the smeared side down, 
upon the plate at this point for one-half to three-fourths minute. Some 
workers, as Pappenheim, use this point, but place the smeared side upward. 

Instead of the copper plate, one may use the ordinary drying oven or a 
Victor Meyer heater. It is the writer's custom to use a copper drying oven 
heated by a gas flame regulated by a thermostat. The slides, with the smeared 
side downward, are placed on a glass plate whose temperature is measured 
by an accurate thermometer. The temperature is allowed to increase gradually 
to about 8o°, from which point it is more quickly raised until the desired 
stage is approximated, when the heating must continue slowly, the final tem- 
perature being maintained for 15 minutes. The best temperature for fixation, 





Fig. 142. — Oven for fixing blood 
films. {Da Costa!) 



THE BLOOD. 475 

in the writer's experience, is no° to 120 for 15 minutes when the staining is to 
be done with eosin-methylene blue or eosin-hemotoxylin, while for the tri-acid 
stain a temperature of 120 to 125 for one and one-half hours should be main- 
tained. Some workers heat to 160 rapidly and then allow the films to cool to 30 , 
when fixation is complete in about 15 minutes. Engel and Cabot recommend, 
in the absence of other equipment, the passing of the smear through the flame 
several times. Such treatment often yields excellent results, but is uncertain 
and requires much experience. It is to be remembered that too rapid changes 
of temperature are to be avoided, as shrinking or splitting of the cells will occur 
under such conditions. So much depends upon proper fixation that a little 
more time spent in obtaining good specimens will shorten the time necessary 
for the future examination. Proper staining depends on proper fixation, 
especially with the tri-acid stain, in which cases the over- or under-heating 
is evident in the color tone of the erythrocytes. 

Chemical Fixation. 
(1). Absolute Alcohol. 

Allow the alcohol to act for five minutes to one hour, or boiling alcohol 
for one minute. The slide is simply covered with the fixative and left for the 
desired period. Much depends upon the stain to be used as to the time neces- 
sary for good fixation. If an alcoholic stain, five minutes is sufficient; if a 
watery or alkaline one, a longer time is essential. After fixation drain off the 
alcohol and allow the residue to evaporate in the air or wash with water and 
dry between sheets of filter-paper. If alcoholic stains are to be used the moist 
specimen may be directly passed through the flame. This fixative is unreliable 
if a study of the neutrophile granules is to be made, but it brings out the nuclear 
structures fairly well. 

(2). Nikiforoff's Method. 

Cover the smear with equal parts of absolute alcohol and ether and allow 
the fixative to act from one-half to two hours. After the fixation allow the 
fixative to evaporate or wash with water and dry. Some workers advocate 
short fixation especially where the malarial organism is to be studied. This 
fixative brings out the degenerations of the red cells in good shape. 

(3). Methyl Alcohol. 

This fixative is used absolutely pure for three to five minutes. It is the 
most generally applicable chemical fixative and gives beautiful specimens. 
If used in combination with stains, as in the Romanowsky methods, it gives 
as perfect preparations as absolute alcohol in one-half hour and brings out the 
neutrophile granulations in better outline. A longer fixation than three to 
five minutes does no harm; and a shorter one, especially if the fixative is the 
solvent for the stain, will give fair results, the outline of the cells being sharper 
the longer the fixative is allowed to act. 

(4). Formalin. 

This fixative may be used as a 1 per cent, solution in 95 per cent, alcohol 
and allowed to act for one minute, as Benario advocates, but the writer has 



476 DIAGNOSTIC METHODS. 

had much better success with the 0.25 per cent, solution in 95 per cent, alcohol 
for one minute, as Futcher and Lazear suggest. Allow the fixative to act 
for one minute, wash in water, and dry between filter-paper. Some writers 
advocate the direct treatment of the fixed smear with the stain without an 
intermediate washing and drying, but I have never found the results as good by 
this method. Instead of the formalin solutions, the vapors may be used by 
placing the specimen under a bell jar with a few drops of 40 per cent, formalin 
and allowing the fixative to act for one to five minutes. The normal staining 
of the cells is not always as clear as could be desired after the use of such a 
fixative. 

A large number of inorganic fixatives have been advised, among them 
being mercuric chlorid, chromic acid and osmic acid, but these fixatives 
are much more apt to produce artefacts than are the others mentioned above. 
While these latter fixatives give good demonstrations of the nuclear structures 
and of mitotic figures, the granules are very imperfect; but chromic acid 
has many advantages as a fixative in the study of the chromatin elements. 

(4). Staining Methods. 

(A). General Considerations. 

Since the work of Witt, we have recognized that the color of an organic 
substance is due to the presence of two definite atomic complexes in the 
molecule of the colored substance. The first of these, the chromophore group, 
is responsible for the chromogenic properties of the substance, while its in- 
fluence as a dye is increased by the presence of the second or auxochromic 
group. The color of the compound is the more intense the more of these 
groups are present. Groups or atoms which intensify the color of the sub- 
stance are called, by Schutze, bathochromes, while those which reduce the 
color are called hypsochromes. 

While the dyes, the so-called anilin dyes, used in hematological work 
are all aromatic derivatives, it must not be assumed that such are alone 
characterized by staining qualities, as many simple aliphatic derivatives show 
a definite color and exert some staining property, depending on the presence 
of the two groups above mentioned. The chief chromophore groups are the 
CO (carbonyl) group, the CS group, CN, HCN, the — N = N — (azo group) 

-N y O 

the yO (azoxy group), NO (nitroso group), — N\ | (nitro group), 

— N' X 

and the — N = SO group. The most important auxochrome groups are the NH a 
and the OH groups, the former being a much more powerful one than the latter. 
These auxochrome or haptophore groups make possible the union of the stain 
with the tissue as a direct salt formation. Substances, which possess a chromo- 
phore group and are colored or intensified by the presence of the auxochrome 
group, are called chromogens. It is, therefore, evident that the effect of an 
auxochrome depends not only upon its own nature, but also upon that of the 
chromogen. Through the introduction of H by means of hydrocarbon radicals, 
new auxochromes are produced and the color becomes intensified, the effect 



THE BLOOD. 477 

being so much the greater the higher the molecular weight of the substituting 
hydrocarbon radical. 

As a large number of colored substances have a tendency to form tautomeric 
compounds, the salt formed by combination with the auxochrome groups 
may have a different constitution from that of the free base or acid. Many 
chromophore groups are also capable of forming salts, but only with strong 
acids or bases. In this formation of salts, by union with the auxochrome 
or chromophore groups, we must remember that halochromia may be seen. 
By this is meant that uncolored or weakly colored substances may combine 
with acids to form salts without the color being due to the presence of a chromo- 
phore group. It is essential that the chromophore group possess a double 
bond of union, as oscillation in one portion of the molecule is thus possible. 
As the chromophoric as well as the auxochromic radicals may have acid or basic 
tendencies, it is manifest that the reaction of the substitution product will 
depend upon the interrelations of the acid and the basic radicals. The best 
dyes are, therefore, obtained by substitution in one direction, that is, by 
accumulating either basic or acid groups separately. 

It is thus seen that we have two general classes of dyes, the acid and 
basic, depending upon the preponderance of the total acid or basic groups in 
the molecule of the dye. It has been shown by Ehrlich that these acid and 
basic dyes, may be so combined that a third one results, showing neither acid 
nor basic properties. This class of dyes, known as neutral dyes, is of the 
greatest importance in hematological work. In them we have not only the 
staining properties due to the original chromophore and auxochrome groups 
of both the acid and basic dyes, but also those due to the union of the component 
groups in the neutral dye. Such dyes are hence called polychrome dyes and 
are usually soluble in an excess of one of the component mother dyes, generally 
the acid one. It must be understood that in speaking of a dye as acid, basic, 
or neutral, we do not refer so much in our staining work to the chemical reaction 
of the dye, but to the portion of the dye to which the staining is due — that is, 
to the specific auxochrome and chromophore groups. 

Among the basic stains we find methyl green, methylene blue, amethyst 
violet, neutral red, dahlia, pyronin, thionin, fuchsin, methyl violet, Bismarck 
brown, alum hematoxylin, and safranin. Most of these stains, depending 
on the strength of their component groups, color the nuclear substance of both 
red and white cells, as also the cytoplasm and certain granules of abnormal 
red and of some normal white cells, the staining being influenced, as Matthews 
has shown, by the alkaline reaction of the tissues. The most important acid 
stains are eosin, acid fuchsin, orange-G, indulin, nigrosin, aurantia, and salts 
of picric acid. These dyes color the red cells and the eosinophile (oxyphile) 
granulations of the leucocytes. The neutral portion of the dyes color the so- 
called neutrophile granules of the leucocytes. 

In the process of staining, it is a question whether we have to do with purely 
chemical processes as Knecht's theory assumes or whether the solid-solution 
theory of Witt or the mechanical theory of surface attraction are accountable 
for the phenomena observed. It is probable that the salts of the dyes are de- 



478 DIAGNOSTIC METHODS. 

composed by the cells and that new compounds result from the union of the 
acid and basic stains and the various chemical entities of the cells. Yet we 
have instances in which the dye is simply stored in the cell without any chemical 
union taking place. We must, however, account for the elective character of 
certain stains by a purely chemical activity, as, for instance, chromatin, which 
undoubtedly consists of nucleinic acid, always takes a basic stain, even though 
a neutral compound is used as the staining agent. It is to be remarked that 
a neutral stain does not color all of the acidophile or basophile substances of 
the cells of the same tint. Thus, eosinophile granules are differently colored 
from the oxyphile hemoglobin of the red cells. The neutral portion of the 
stain has nothing to do with the staining process beyond the coloring of the 
neutrophile substances in the protoplasm of certain leucocytes. 

In the selection of a stain for general hematological work, it is necessary 
to use a compound stain or, at least, two simple stains, one after the other. 
A single simple stain colors only a few of the elements and affords no general 
idea of the blood picture. Indeed, to obtain definite conceptions of the finer 
structure of the blood, it is necessary to study several slides stained by different 
methods. It is usual to select for routine work a stain which will reveal the 
greatest amount of information regarding the histological characteristics of 
the blood. This is the so-called panoptic staining and is to-day generally 
accomplished by the use of the various forms of neutral stains. In making the 
stains for one's self or in buying them in the market, one must be certain that 
only chemically pure pigments are used and that the solution is made accord- 
ing to the formula with the purest solvent obtainable. It will need only one 
experience with a poorly made stain to convince the worker of the importance 
of this detail. 

(B). Methods of Staining. 

In his work the author uses, for routine purposes, the Wright's stain, 
the eosin-methylene blue, and eosin-hematoxylin stains. These stains have 
the advantage of giving clear-cut pictures of practically all of the important 
blood elements along with simplicity of technic. As it is not always possible 
to secure or even to make a Wright or an Ehrlich triple stain which will 
give reliable results under all circumstances, the writer feels that the general 
worker would better use the eosin-methylene blue stain for his daily work. 
The many modifications of the Romanowsky stain have their advocates, and it 
must be said that they yield reliable results when they are working properly, 
but no one can say when they will go wrong or how to make them work right 
when once they do give poor results. 

Eosin-Methylene Blue. 

A number of methods of using these two simple stains have been advocated 
by such workers as Chenzinsky, Ehrlich and Lazarus, von Willebrand, Plehn, 
Aldehoff, and Gabritschewsky, but the writer has found the method advocated 
by Mullern x to be the most generally applicable and, if properly applied, the 
most reliable modification. By it we are able to stain all of the blood elements, 
1 Grundriss der klinischen Blutuntersuchung. Wien, 1909. 






THE BLOOD. 479 

including the neutrophile granules and obtain, thus, a panoptic picture whose 
findings are not excelled by those of the various modifications of the Roman- 
owsky stain. 

Technic. 

(a) Fixation of the smear in pure methyl alcohol for three minutes, (b) 
Preliminary staining in 1/2 per cent, alcoholic (70 percent.) solution of Grubler's 
"french pure" eosin from three to five minutes, (c) Wash in distilled water 
and dry between filter-paper, (d) Lay the slide in a carefully measured 
and well-mixed solution of 20 drops of 1/4 per cent, aqueous solution of 
methylene blue (B. pat.) and ten drops of the above eosin solution for one- 
half to one minute, (e) Wash quickly and briefly with distilled water and 
dry at once between filter-paper or over the flame. (/) Mount if desired in 
Canada balsam or examine directly with a high-power lens. 

This stain shows the red cells and the eosinophile granules of the leuco- 
cytes of a bright red tone, the neutrophile granules pink to bright red (distin- 
guished from the eosinophiles by their smaller size), the nuclei, mast-cell 
granules, bodies of the lymphocytes, platelets, malarial organisms, trypanosomes, 
and filaria varying shades of blue. The preliminary staining with eosin serves 
the purpose of bringing out the neutrophile granules more clearly while the 
basophile granulations are of course unaffected by such treatment. It is 
probable that the later staining with the eosin-methylene blue mixture has the 
same characteristics as the neutral stains to be mentioned later. While this 
stain has the advantage of simplicity, reliability, and panoptic power of staining, 
it is somewhat inferior to other stains in bringing out some forms of the malarial 
organism, owing to its lack of chromatin staining qualities. The writer has 
found that the eosin and methylene blue above mentioned are the best to use in 
this process and that Turk's advice, regarding the use of fairly fresh eosin 
solutions and of old methylene-blue solutions, is warranted. The blood prepa- 
rations should not be over a few days old to show the best pictures, as those over 
a week old may show a diffuse plasma staining and the neutrophile granules not 
clearly differentiated. If the nuclei do not stain well with the methylene blue 
as many writers claim, make a second preparation fixing somewhat longer 
in methyl alcohol. By following out these precautions the worker will be 
rewarded with beautiful specimens. This method is to be recommended for 
all routine examinations. 

Eosin Hematoxylin. 

This stain is especially important in cases in which the nuclear structures 
are to be studied. l It stains the nuclei beautifully, showing their finer structure, 
karyokinetic figures, and pycnotic qualities, as well as the basophile granules 
of both red and white cells. The solutions required are: (1) 1/2 per cent. 
Grubler's blood eosin in 70 per cent, alcohol. We may use, with equally good 
results, the eosin mentioned in the previous method. (2) Delafield's hema- 
toxylin, the formula of which is 

1 See Williams (Jour. Am. Med. Assn., 1913, LXI, 1627), who describes a modification of 
this stain which he calls "invariable." 



480 DIAGNOSTIC METHODS. 

Hematoxylin crystals, 4 grams. 

Alcohol (absolute), 25 c.c. 

Ammonium-alum crystals, C. P., 52 grams. 
Distilled water, 400 c.c. 

Glycerin, C. P., 100 c.c. 

Methyl alcohol, C. P., 100 c.c. 

Rub up the hematoxylin crystals with the alcohol until they are dissolved 
and place the solution in a loosely corked glass bottle, allowing it to stand 
exposed to the light for four days. Dissolve the ammonium-alum in the water 
and allow it to stand exposed in the same way for four days. At the end of 
this time mix the two solutions, shake thoroughly, and filter at the end of three 
hours. Add the glycerin and methyl alcohol to the filtrate and allow this to 
stand overnight. Filter the mixture, place it in a clear bottle, and allow it 
to ripen, exposed to the light for six weeks, when it is ready for use. 

Technic. 

Stain the specimen with the eosin solution for one-half minute, and wash 
in water. Without drying place the slide in the hematoxylin solution for 
one to three minutes, the time varying with the particular stain and with the 
experience of the worker. Wash with water, dry, and mount. This stain 
does not give as good results as does the former method, but is to be especially 
recommended when the nuclear structures are to be studied. 

Ehrlich's Triple Stain. 

In the literature of hematology we find the expressions "triacid" 
and " triple stain" used synonymously. The triacid stain, as originated 
by Ehrlich, was a mixture of equal parts of saturated solutions of indulin, 
nigrosin, and aurantia, and was used to differentiate the eosinophile granules. 
When used synonymously in these days, the triple stain is always meant. 
The composition of this latter stain is as follows, made up of two acid and 
one basic stain: 

Saturated watery solution of orange- G, 

Saturated watery solution of acid fuschin, 

Distilled water, 

Alcohol (absolute), 

Saturated watery solution of methyl green 00, 

Alcohol (absolute), 

Glycerin, 

The pigments must be chemically pure and the solutions must be added 
in the order named, the methyl-green solution being added drop by drop with 
constant stirring. 

This stain is difficultly prepared, being usually a failure. The writer 
would, therefore, advise the worker to obtain it already made; and even then 
it may not prove satisfactory. The correct stain should have a russet-brown 
color and should not show any trace of a precipitate. It improves up to a 



3-14 


c.c. 


6-7 


c.c 


i5 


c.c. 


15 


c.c. 


12.5 


c.c. 


10 


c.c. 


10 


c.c. 



THE BLOOD. 48 I 

certain point with age but, after a time, precipitates invariably occur, making 
it useless. It should never be filtered or shaken, the solution to be used being 
taken from the center of the bottle with a dropping pipet. 

Technic. 

The smear should be fixed by heat as previously described. After proper 
fixation, cover the smear with the stain and allow it to act for one to ten minutes 
as the experience of the worker with the particular stain may indicate. Wash 
with distilled water, dry, and mount. One advantage of this stain is that 
it cannot overstain, those films appearing so being underheated, while those 
understained are overheated. 

This stain shows the red cells of a buff or orange color, without the 
slightest shade of red (a reddish tint is given with underfixed specimens while 
a yellow tone is shown by those over-heated), the nuclei of the leucocytes 
a dark green, those of the normoblasts black, the neutrophile granules a lilac 
color (though some occasionally show a reddish tinge), and the eosinophile 
granules of a crimson tone. 

This stain was introduced as specific for the neutrophile granules, but its 
disadvantages are too numerous to warrant its recommendation as a routine 
stain. It is a poor nuclear stain, does not show the structure of the normal 
mononuclear leucocytes, does not stain the basophile granules, nor the malarial 
or other parasites. For a reliable preparation, showing those features for 
which it is especially adapted, a proper fixation is an absolute essential. 

On account of the lack of nuclear staining with the triple stain, Pappen- 
heim has substituted methylene blue or methylene azure for the methyl green, 
and eosin for the acid fuchsin. The writer has had no experience with this 
modification and cannot, therefore, speak regarding its value. 

Polychrome Methylene-blue-eosin Stains. 

These stains are very numerous, each having its advocates. They are 
easy to use, contain a reliable fixative, and give satisfactory results, but are 
not always obtainable or easily prepared.- They are the stains which give, 
perhaps, the best panoptic results and are especially serviceable in the study 
of the malarial organism and other parasites as many of them contain chroma- 
tin-staining elements. The granulations of the leucocytes are not as well 
marked in all cases as they should be, so that for a complete study of the 
various types of granules in the cells several stains would better be used. 

Romanowsky had found that the addition of a watery eosin solution to 
an aqueous methylene-blue solution, until an insoluble precipitate began to 
form, gave rise to new staining properties of the solutions, in the sense that 
the chromatin substance of malarial organisms was stained a beautiful red. 
The specific staining properties of this mixture were later found to be due 
not to a combination of eosin and methylene blue, per se, but rather to the 
formation of a new compound between eosin and an impurity or decomposition 
product in the methylene blue, namely, methylene azure. Jenner in his 
stain makes use of a methyl-alcohol solution of the isolated precipitate, the 
31 



482 DIAGNOSTIC METHODS. 

eosinate of methylene blue, which lacks the red chromatin staining element. 
The same may be said of the May-Griinwald stain. 

In making up these polychrome stains it is not general to use the pure 
methylene azure and eosin, but rather solutions of methylene blue containing 
a variable amount of the methylene azure to which eosin is added. Recently 
Wilson, 1 in a careful study of the methylene-blue-eosin combinations, has 
shown that very little methylene azure and methylene violet exist in the stains 
as commonly employed. He finds evidences of at least four staining bodies in 
such mixtures, namely, the eosinate of methylene blue, eosinate of methylene 
violet, eosinate of methylene azure, and eosinate of thionin. 

Wright's Stain. 

This stain is a much improved modification of the Leishman stain, 2 as its 
method of preparation requires but a few hours and as the resulting dye is 
somewhat more panoptic than is that made by the Leishman method. 

Preparation. 

To a 0.5 per cent, aqueous solution of sodium bicarbonate add methylene 
blue (B. X. or "medicinally pure") in the proportion of 1 gm. of the dye to 
each 100 c.c. of the solution. Heat the mixture in a steam sterilizer at ioo° C. 
for one full hour, counting the time after the sterilizer has become thoroughly 
heated. The mixture is to be contained in a flask, or flasks, of such a size and 
shape that it forms a layer not more than 6 cm. deep. After heating, allow the 
mixture to cool, placing the flask in cold water if desired, and then filter it to 
remove the precipitate which has formed in it. It should, when cold, have a 
deep purple-red color when viewed in a thin layer by transmitted yellowish 
artificial light. It does not show this color while it is warm. To each 100 c.c. 
of the filtered mixture add 500 c.c. of a 0.1 per cent, aqueous solution of "yellow- 
ish, water-soluble" eosin and mix thoroughly. Collect the abundant precipi- 
tate, which immediately appears, on a filter. When the precipitate is dry, dis- 
solve it in methyl alcohol (Merck's "reagent") in the proportion of 0.1 gm. to 
60 c.c. of the alcohol. In order to facilitate solution, the precipitate is to be 
rubbed up with the alcohol in a porcelain dish or mortar with a spatula or pestle. 
This alcoholic solution of the precipitate is the staining fluid. It should be 
kept in a well-stoppered bottle because of the volatility of the alcohol. 3 

Technic. 

Cover the film with a noted quantity of the staining fluid by means of a 
medicine dropper. After one minute add to the staining fluid on the film the 
same quantity of distilled water by means of the medicine dropper and allow 

1 Jour. Exper. Med., 1907, IX, 645: 

2 Leishman Stain. Prepare a 1 per cent, aqueous solution of medicinal methylene blue 
(Griibler) and add 0.5 per cent, sodium carbonate; heat for 12 hours at 65 C. and keep for 
10 days at room temperature. Prepare a 0.1 per cent, aqueous solution of "yellowish, water- 
soluble" eosin. Mix equal volumes of the two solutions and leave for 10 to 20 hours, shaking 
the mixture at frequent intervals. Filter, wash the precipitate with distilled water until the 
washings are a pale blue, dry on the filter and powder the dried residue. The staining solu- 
tion is prepared by dissolving 0.2 gram of this powder in 10 c.c. of methyl alcohol. This 
stain is used precisely as the Wright stain, the blood-picture being very similar. 



Jour. Am. Med. Assn., 19 10, LV, 1979. 






THE BLOOD. 483 

the mixture to remain for two or three minutes, according to the intensity of the 
staining desired. A longer period of staining may produce a precipitate. 
Eosinophilic granules are best brought out by a short period of staining. The 
quantity of the diluted fluid on the preparation should not be so large that some 
of it runs off. Wash the preparation in water for thirty seconds or until the 
thinner portions of the film become yellow or pink in color. The experience of 
the worker may be such as to demand longer decolorization with any particular 
specimen. When the desired degree of differentiation is reached, dry the speci- 
men quickly between filter-paper, mount if desired, and examine first with 
the low-power lens to observe the staining effects, and then with the high-power 
lens for the more minute study. When searching for malarial parasites, the 
decolorization would better be of short duration, as the chromatin suffers to a 
great extent in this process. 

This stain shows the red cells colored either orange or pink (depending 
on the time of decolorization), nuclei of leucocytes blue or dark lilac, neutro- 
phile granules lilac, eosinophile granules red or pink, fine basophile granules 
deep blue, large mast-cell granules purple, protoplasm of the lymphocytes 
robin's-egg blue, blood-plates deep blue or purple, bacteria blue, malarial 
and other parasites blue, the chromatin element varying from lilac to ruby- 
red to black. Polychromatophilia and granular degenerations are well 
shown, the granules being blue. This stain is very useful in studying lympho- 
cytes, mast cells, blood-plaques, and the finer structure of the malarial organism; 
but the leucocytic granules, at times, are not sufficiently differentiated. 

Giemsa Stain. 

Giemsa has shown that the complicated methods of preparing the dyes 
may be dispensed with by the use of the pure staining substance extracted from 
the polychrome methylene blue, namely, the methylene azure. If this pure 
pigment be used the stain becomes a pure chromatin one. It is advisable 
therefore, to use Grubler's azure II, a combination of equal parts of methylene 
azure and medicinal methylene blue. The formula of the Giemsa stain is as 
follows : 

Azur II-Eosin, 3.0 grams. 

Azur II, 0.8 gram. 

Glycerin (C. P.), I 25-o grams. 

Methyl alcohol (C. P.), 375.0 grams. 

Grind up the dyes in the alcohol and then add the glycerin. 

Technic. 

Fix the films in methyl alcohol, and stain for fifteen minutes in a mixture 
of 15 drops of the stain to 10 c.c. of distilled water. If necessary, a trace of 
sodium carbonate may be added to the water to intensify the basic stains. 
Wash in water, dry, and mount. It is to be remembered that oil of cedar 
will bleach these specimens quite rapidly, so that it is advisable, if the films are 
to be kept, to stain them upon the slides so that mounting becomes unnecessary. 
Strong light should not be allowed to act on these stained films for any length 



484 DIAGNOSTIC METHODS. 

of time as the chromogen stain fades rather quickly under these conditions. 
The various elements are stained as with the Wright stain, but the neutrophile 
granules are often not well defined. 

Pappenheim's Stain. 

Pappenheim (Technik der klin. Blutuntersuchungen, Berlin, 1911; Folia 
Haemat, 1912, XIII, 339) advocates as the best and most serviceable stain 
the combination of the Giemsa stain as follows: Fix the blood film by covering 
it with May-Grunwald stain (Wright's answers as well) for 3 minutes, add to 
this stain on the slide an equal amount of distilled water and allow it to act for 
1 minute. Pour off the fluid but do not wash. Now cover the stained film 
with dilute Giemsa solution (15 drops of stain to 10 c.c. of distilled water) and 
allow to act for 15 minutes. Wash thoroughly and dry, but not over the flame. 
Mount, if desired, in neutral Canada balsam. The result is extremely panoptic. 
The nuclei stain violet. The lymphocytes, monocytes and undifferentiated 
lymphoidocytes have a delicate blue cytoplasm. The mast-cell granulations 
are ultramarine blue. Eosinophiles have brick-red granulations and the 
neutrophiles a rose-colored cytoplasm, while their granules are indefinite. 

Specific Stains for Malarial Organisms. 

While the above stains all give good results with the malarial organisms, 
enabling one to make a diagnosis from an examination of the smear, yet certain 
other stains are often of advantage in that little else is stained and thus the 
confusion arising from indefinite staining is obviated. 

Thionin Stain (Futcher and Lazear). 

Add to 100 c.c. of 2 per cent, carbolic acid 20 c.c. of a saturated solution 
of thionin (Lauth's violet) in 50 per cent, alcohol, and allow this mixture 
to ripen for a few days. Fix the specimen by the formalin method given above 
and stain the smear for 10 to 15 seconds. Wash in water, dry, and mount. 
These preparations do not keep indefinitely, usually fading within a year. 

The plasmodia are shown as deep purple, irregular masses enclosed in 
the faint green red cells. The hyaline forms show as reddish-violet ring-like 
bodies. The basophile granules and the nuclei are the only other elements 
showing any particular staining qualities. 

Nocht's Stain. 

This is essentially a Romano wsky stain and is made as follows: Add 
two or three drops of 1 per cent, aqueous eosin solution to 2 c.c. of water. 
To this diluted eosin solution polychrome methylene blue is added, drop by 
drop, until the red color of the eosin is only faintly visible (the polychrome 
methylene blue is a 1 per cent, solution of methylene blue chemically changed 
by heating with 1/2 per cent, sodium carbonate solution for two days at 55 C). 

Technic. 

Fix the film in methyl or ethyl alcohol and lay it face downward in the 
above mixture for five to ten minutes. Wash with water, dry and examine. 
The picture is the same given by Wright's stain, except that the nuclear trans- 



THE BLOOD. 485 

formations and the chromatin substance are better differentiated, being stained 
a bright red color. 

The many other stains for the examination of fixed specimens will be passed 
over, as the writer finds the above stains applicable to practically all routine 
.work. Many writers prefer different stains from those mentioned, but these 
stains do not seem to have any advantages which would warrant their use to 
the exclusion of those given above. Special stains used for bringing out 
certain granules and deformities will be discussed under later headings. 

Vital Staining. 

Although the examination of stained specimens of the blood is usually 
made with the dried and fixed smear, excellent results obtain when the fresh 
blood is stained without previous fixation. It is true that a " vital" staining 
of the blood-cells does not actually take place, as the dyes are decolorized 
by the reducing and oxidizing action of the living cells. However, a "post 
vital" staining, that is the staining of whole cells or portions of the cells, after 
their removal from the circulation and before the death of the cell results, 
may be accomplished in several ways. 

We may either add to the fresh drop of blood a few crystals of the stain, 
as advised by Arnold, and note the staining of certain leucocytic granules 
and nuclei and recticular structure of many erythrocytes; or we may first dry 
a staining solution upon the slide, cover this dry stain with a drop of fresh 
blood, adjust the cover-glass, and seal this to the slide with wax. Some 
workers prefer to use the hanging-drop slide in the preparation of these fresh 
specimens. 

The stains which may be used for vital staining of the nuclei, granules, and 
plates are methylene blue, toluidin blue, thionin, neutral violet, Capri blue, Nile 
blue, brilliant- cresyl blue, Janus green, and paraphenyl blue. Of the proto- 
plasmic stains we have fuchsin, acridin red, pyronin, safranin, and neutral red. 

While the results derived from this method of staining are not as numerous 
as those of the more usual method, yet it affords much valuable information 
regarding the vital properties of the cells and regarding the normal structure 
and the circulatory changes of the cells. 1 This method has been used extensively 
by Ito, Rosin and Bibergeil, Levaditi, Cesaris-Demel, and Pappenheim. 
Their results lead us to assume that further study will give us much valuable 
information concerning details of structure both of the cell and of the nucleus. 
It may be possible by this method to differentiate between certain forms of 
degeneration of the cell which are now known only indefinitely under the names 
of metachromatic and polychromatic staining. 2 

1 See Luzzatto and Ravenna (Folia. Haemat, 1912, XIII, 102) for a discussion of the 
numerical relations of the granular and non-granular erythrocytes as shown by this method. 

2 See Ross (Induced Cell-reproduction and Cancer, Philadelphia, 1911, and London, 191 2) 
for the use of agar films containing stains and activating salts (auxetics) in this connection. 
See, also, Anitschkow, Med. Klin., 1914, X, 465; Barbaro, Gazz. d. osp., 1914, XXXV, 681; 
von Mollendorff, Deutsch. med. Wchnschr., 1914, XL, 1839; Hober, Biochem. Ztschr., 1914, 
LXVII, 420; Lindborn, Nord. Med. Arch., 1914, XLVII, 1; Evans and Schulemann, Science, 
1914, XXXIX, 443; Folia Haemat., 1915; XIX (I), 207; Rubino, Rif. Med., 1915, XXXI, 533; 
Traube, Biochem., Ztschr., 1915, LXIX, 309. 



486 



DIAGNOSTIC METHODS. 



(5). Erythrocytes. 
(A). Appearance and Structure. 
In fresh, normal blood the red cells, or erythrocytes, appear as thin, 
flattened, homogeneous, biconcave, nonnucleated, discoid bodies with a 
sharply denned regular outline and a clear semitransparent center. In some 
cases they may appear distinctly cup- or bell-shaped. The cells show, when 
examined singly, a pale greenish-yellow color but, when more thickly grouped, 
exhibit a reddish tint. The degree of color in these cells depends upon their 
hemoglobin content, the clear central area becoming larger and the entire cell 
becoming paler as the hemoglobin decreases. The loss of hemoglobin may be 
so great as to lead to the formation of the so-called " pessary" form, in which 
only the periphery of the cell is apparent. This central pale area varies much 
in individual cells and is not at all evident in those which are flattened out. In 
dry specimens, if thinly spread, the cells are circular, the normal biconcavity 




Fig. 143.— Normal blood showing rouleaux formation and fibrin network. (Da Costa.) 

is obliterated, and a uniform stain is observed. If the smear be thick the 
central pale area is observed. 

In chlorosis and secondary anemias, the color of the cells is usually uni- 
formly paler than normally, while in pernicious anemias the color may be even 
deeper than normal. These are the usual but not invariable pathologic 
findings. In malarial conditions, discolored cells are often observed in the 
fresh specimens, the bronzed or " brassy" tone often drawing attention to the 
presence of a parasite of the estivoautumnal type or of the quartan form. 

The red cells show a marked tendency to cohere to one another in more 
or less regularly arranged piles, forming long rows (rouleaux), like rolls of 
coin piled up face to face. The exact cause of this phenomenon is unknown, 
but it may be dependent on the presence of the fatty membrane surrounding the 



THE BLOOD. 487 

cell, as Peskind's findings show that the red cells are enveloped by a layer com- 
posed of lecithin, cholesterin, and a nucleoprotein. 1 In certain pathologic condi- 
tions this normal rouleaux formation is increased and in certain ones is de- 
creased. The diminished rouleaux formation is observed in conditions associated 
with increased viscosity of the cells, as observed in most inflammatory diseases 
and in the anemias due to malignant disease. This hyperviscosity of the 
cells has not at present much clinical significance, but may be shown to be of 
importance when our knowledge concerning the normal viscosity of the blood 
becomes more extensive. 

The structure of the erythrocytes is still an unsettled point in hematology. 
Neither membrane nor stroma have been fully demonstrated yet after the 
hemoglobin has been removed from the cells by hemolysis, a stroma may 
be definitely seen which could hardly be called an artefact. Schafer assumes 
that the hemoglobin is held in firm combination by chemical union with other 
albuminous constituents of the cell and supported by a stroma similar to that 
of Peskind's outer membrane. That some sort of an outer membrane does 
exist would seem to be proven by the experiments on hemolysis which have 
shown that many substances penetrate the cell producing hemolysis, while 
others in the same concentration have no such effect. It is hard to believe that 
a selective vital activity is at work here, as the results follow too closely the laws 
of physical chemistry as applied to diffusion and osmosis. The corpuscles are 
very elastic and contractile so that rapid and marked temporary distortions of 
shape are possible under the influence of variations in the composition of the 
circulating plasma. 

As the blood dries various changes in the appearance of the red cells are 
observed. These changes, known as crenation, are due to the evaporation 
of water and depend upon the quantity of air which comes in contact with the 
specimen as well as upon the length of time this influence acts. "The develop- 
ment of one or more small, bright, highly refractile spots in the body of the 
cell and a slight indentation of the periphery of the cell are the most conspicuous 
indications of beginning crenation. As the process goes on, more and more 
of these hyaline points develop, until finally the whole surface of the corpuscle 
becomes thickly studded with glistening bead-like spines. As the stroma 
becomes drier and drier, its typical biconcavity and sharply cut outline are lost, 
contracting strands of the stroma are seen to extend from point to point among 
the beaded projections, the periphery of the cell changes to a cogged rim, and 
finally the cell becomes shrunken and shriveled up into a small, many-starred 
asterisk. Some of the erythrocytes become fragmented and small bits of their 
stroma are observed to break off and float through the plasma. Others 
become progressively paler and paler, as the hemoglobin is dissolved out, 
until complete decoloration occurs. Still others become distorted into designs 
of every conceivable shape so that their resemblance to the normal cell becomes 
more remote" (Da Costa). These changes must not be confused with those 
occurring as a result of pathological changes. Crenation is often induced 
more rapidly than in normal blood, in the blood of persons suffering from 
1 See Wiltshire, Jour. Path, and Bacterid., 1913, XVII, 282. 



DIAGNOSTIC METHODS. 

acute infection and from chronic diseases. True ameboid movement of the 
red cells is sometimes seen as a result of a high-grade anemia. 1 

(B). Size and Shape. 

The average diameter of these normal human erythrocytes (normo- 
cytes) is 7.5 jjl, the normal variations being between 6 and 9 u. (a micromiilimeter, 
1 / 1000 of a millimeter) . The size varies depending on the method of preparing 
the specimen and also upon the osmotic tension of the plasma. Although 
dwarf and giant cells may occur to a slight degree at all ages, the normal infant 
blood shows these variations more markedly (3.3 to 10.3 //, according to 
Hayem). According to Hamburger, the cells are slightly larger in the venous 
than in arterial blood. Gram states that the size of these cells varies with 
climatic conditions, being greater in those of the northern cooler countries 
than in those of the southern warmer climates. The variations in the size 
of the red cells is very slight in the different sexes. 

Pathologically, variation in the size of these cells is a common and 
important feature. Generally speaking, variations in the cellular size indicate 
a severe and chronic anemia, while in the more mild acute forms of anemia 
such variations are unusual. The average size is said to be increased in jaundice, 
cholera, lead-poisoning, leukemia, congenital heart disease, and cretinism. 

Types of Pathological Erythrocytes. 

Variation in the normal size of the red cells is indicated by the term 
anisocytosis. This term does not include the misshappen red cells which 
appear in the blood as a result of degeneration or of mechanical injury and to 
which are given the name of poikilocytes. 

Microcytes. 

These are cells under the normal size, the variations being between 1 a 
and 6 fi, the usual representatives being about 3.5 /i. It cannot be stated 
at present whether these cells are mere schistocytes (fragments of larger cells) 
or are perfect cells of degenerative origin. They occur normally in the blood 
of embryos and infants, but are rare in that of the adult except in pathological 
conditions. It is certainly true that these undersized cells may arise from 
purely physical causes, as a result of increased osmotic pressure of the plasma 
as well as from division of undersized mother cells. This latter phase is made 
possible by the appearance, especially in pernicious anemia, of nucleated reds 
of corresponding size. 

Pathologically, these smaller cells are observed in all severe anemias. 
As a rule, they stain deeply and evenly, but in some cases of pernicious anemia 
these cells are deficient in hemoglobin and show irregular staining, yet many 
of them may have an increased hemoglobin content. Occasionally, but not 
invariably, we find these undersized cells in chlorosis, in which the hemoglobin 
is deficient. At times these cells may show polychromatophilia, but this is 
not the rule. According to Tallqvist, an increase in the number of these micro- 
cytes (microcytosis) is an indication of rapid destruction of blood. 

1 See Krizenecky, Ztschr. f. alls. Physiol., roiq, XVII, 1. 



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Types of Red Cells. 
(Wright's Stain. Zeiss Ocular. 4, Oil Immersion Objective.) 

1 — Normal Cells. 

2-3-4 — Normal Cells as seen with different Focus. 

5 — So-called "Pessary Form." 

6-7-8 — polychromatophhic cells. 

9 Macrocyte. 

io-12-i3 — poikilocytes. 

1 1 — poikiloblast. 

14-15 — Punctate Basophilla. in Red Cells. 

16-17 — Normoblasts. 

18 — Normoblast with Pycnotic Nucleus. 

19 — Punctated Normoblast. 

2 0-2 3 — Megalob lasts. 

24 — Gigantoblast. 



THE BLOOD. 489 

Macrocytes. 

These are cells above the normal size, the variations being between 10 
and 20 fi. Those cells from 9 to 12 fx are called macrocytes; those between 
12 and 16 /jl are known as megalocytes; while those above 16 jj. are termed 
gigantocytes. 

These cells are of regular shape, of even staining qualities, and generally 
without a well-defined central clear area. The larger size of these cells may 
be partly due to the swelling incident to a lowered osmotic tension of the blood, 
but more probably this increase in size is traceable to the origin of the macrocytes 
from the large nucleated reds of the bone-marrow. These cells may show 
an excess or deficiency in hemoglobin, the former characteristic being observed 
in the primary pernicious anemias, while the latter is evident in the secondary 
forms. Morris and Thayer 1 report amebeid movements in these cells. 

Pathologically, the presence of these various forms of macrocytes, giving- 
rise to the condition of macrocytosis, indicates a severe and unusually chronic 
anemia. They are most frequently seen in pernicious anemia, in which the 
largest cells are sometimes the darkest and some of the microcytes are exceed- 
ingly pale. These large cells do occur, however, in leukemia, cholemia, and 
chlorosis, being frequently pale or "chlorotic" and "dropsical." These 
"dropsical" cells are not sufficiently numerous, however, to change the volume 
index, as Capps has shown, of a secondary anemia to one shown in true per- 
nicious anemia. 

Poikilocytes. 

These are misshapen red cells of large or of small size, the varieties of 
such deformities being numerous. The presence of poikilocytes in the blood 
is known as poikilocytosis (first described by Damon) and is closely related 
to crenation as in both cases the cells may be similarly misshapen. The former 
is a pathologic process demonstrable the moment the blood is taken, while the 
latter is a physiological process appearing only after the blood has been in 
contact with air for some time. 

Poikilocytes arise in several ways. First, faulty technic, especially 
pressure on the cover-glass of the fresh specimen, will give rise to fragmentation 
of some of the corpuscles into small spherical masses, dagger-shaped bodies, 
and small elongated rods (pseudo bacilli of Hayem). Such fragmentation is 
indicative of lowered vitality and feeble powers of resistance of the cells. If 
the cover-glass be moved after the cells have spread, a large number of them 
will be distorted into oval or pear-shaped forms, the long axes of which usually 
point in the same direction. Secondly, true poikilocytes, or cells misshapen 
while in the circulation, are probably due to ameboid motion of a portion or 
the whole of a cell or to alterations in the plasma. These misshapen cells are 
usually pear-shaped with a budded projection at one or more poles. Cells 
may be seen which resemble a tennis-racket, a kidney, tomahawk-blade, 
dumb-bell, or anvil, while oval forms are especially observed in pernicious 
anemia and are considered by Cabot of diagnostic importance. Poikilocytosis 

1 Arch. Int. Med., 1911, VIII, 581. 



490 • DIAGNOSTIC METHODS. 

is an indication of severe anemia with degenerative changes in the red cells; 
although it is not characteristic of any single disease, it is found more fre- 
quently in pernicious anemia and leukemia. 

(C). Nucleation. 
Nucleated red cells may be considered pathological at any period of 
extrauterine life, although they are usually found in the blood of the child 
during the first few days of life. These nucleated reds are always found in 
the bone-marrow, the normal and large forms being quite distinctive. The 
large form is the oldest and gives rise by cell division to the smaller cell or the 
normoblast. It is probably true that the nonnucleated red cells are derived 
from the nucleated form, but the denucleation takes place before the normal 
cells reach the blood. Just how we are to explain the disappearance of all 
trace of the nucleus from the normal erythrocyte is a question, but the general 
concensus of opinion seems to be that the nuclear material gradually fades 
within the cell, although some slight evidence of extrusion of the nucleus can 
be advanced: Although our ordinary methods show no such evidence, King 1 
has recently introduced a method by which he demonstrates stainable remnants 
of a nucleus in practically every red cell in the normal adult blood. 

Normoblasts (Trachyochromatic Ery throblasts) . 

These are nucleated red cells similar in size, shape, and color to the 
normocytes. They do not usually show a biconcave form and do not unite in 
rouleaux. The protoplasm of the cell is usually regular in outline, stains more 
intensely than does that of the normocyte and frequently shows evidence of 
polychromatophilia, although it is normally orthochromatic. In myeloid 
leukemia, cells are frequently observed in which the protoplasm presents 
a ragged outline and may even, in some cases, be so degenerated as to show 
only a small fragment attached to the nucleus. These latter cells are practically 
always polychromatophilic. This type of small cell might be called a microblast, 
which is the rarest form of erythroblast and corresponds in size to the microcyte. 
Moreover, we may find such cells or fragments of cells attached to a nucleus, 
such cells corresponding in size and shape to the poikilocytes and being termed 
poikiloblasts. 

The nucleus of the mature normoblast (Howell's mature nucleated red) 
has a diameter of about one- third that of the cell, is densely stained, homogeneous, 
sharply defined, spheroidal in shape, and without any decided chromatin net- 
work (the so-called pycnotic nucleus). It is situated rather toward the per- 
iphery than in the central portion of the cell and is surrounded by a clear zone 
shading off into the cellular protoplasm. Occasionally the nucleus is observed 
resting upon a margin of the red cell or even extruded entirely from it probably 
as a result of degeneration of the surrounding protoplasm (Pappenheim) . 
These nuclei often show amitotic figures, being subdivided into two or more 
lobes or fragments which may be connected by strands of chromatin. These 
mature forms may show all gradations in which the chromatin network becomes 
more and more evident, until we reach the very immature forms (Howell's 
1 Jour. Med. Research, 1911, XXIV, 91. 



THE BLOOD. 49I 

immature nucleated reds). These latter cells are somewhat larger than 
the mature forms and are somewhat lighter in color; the nucleus is relatively 
larger and is composed of delicate faintly basic chromatin fibers radially 
arranged, and frequently showing mitotic figures. 

These two types of cell are the forerunners of the normocyte and their 
appearance in the adult blood is indicative of increased activity of the hemato- 
poietic organs, especially of the bone-marrow, either as a result of a poor condi- 
tion of the blood itself or as a direct disease of the blood-forming organ. These 
cells are most commonly seen in the milder forms of anemia, chlorosis, and acute 
anemia from hemorrhage, inanition, or organic disease. In the severe types of 
anemia they are constantly met with and are usually associated with the larger 
megaloblasts. During the course of severe anemia, especially in chlorosis, there 
obtains a periodic increase of normoblasts and of leucocytes lasting several 
days. This is followed by a marked increase in the normal number of red cells, 
giving rise to the condition described by von Noorden as a " blood crisis. " 
The normoblasts disappear, the blood count falls off, and a second crisis may 
obtain. This condition is considered as a transitory attempt on the part of 
the bone-marrow to regenerate the blood. Such a crisis is not always a sign 
of improvement, but it does demonstrate that the type of blood formation is 
becoming physiological and that recovery may follow. 

Megaloblasts (Amblyochromatic Erythroblasts) . 

These are cells of larger than normal dimensions, corresponding in size 
to the macrocytes and varying from 9 to 20 microns in diameter, if exceeding 
20 microns they are known as the gigantoblasts of Ehrlich. Occasionally 
some cells are seen which are no larger than a normoblast, but in which the 
nucleus shows definite characteristics which should enable one to classify the 
cell as a megaloblast (Pappenheim) . 

The protoplasm of the megaloblast often appears swollen and enlarged 
(dropsical). The cell is usually circular or oval, but it is easily deformed, 
giving rise to irregular-shaped bodies. Although it usually contains an excess 
of hemoglobin, it may show a deficiency. It is usually polychromatophilic, 
the shade of cellular staining varying from yellow to purple. These various 
color tones may not be regular, but may be varied by tintings of almost any 
shade in the same cell. 

The nucleus of the megaloblast is very large, varying between 6 and 10 
microns. This may be situated either centrally or somewhat peripherally. 
It shows a great variety of forms appearing as a vesicular body with intra- 
nuclear network and nodal thickening, but without nucleoli. It may rarely be 
pycnotic, may show mitoses or many stages of karyorrhexis with fragmentation, 
vacuolation, fading of the segments of the nuclei, as well as minute subdivisions 
into fine basic staining particles widely scattered in the cell (Ewing).. It 
is frequently poorly defined and shows feeble basic staining qualities. It may 
be sharply differentiated from the body of the cell by a distinct white margin 
which is thrown into relief by the deeper staining of the nuclear and cell-sub- 
stances. Occasionally the nucleus is overlooked owing to the polychromato- 
philic properties of the cell, which do not always permit of clear differentiation 



492 DIAGNOSTIC METHODS. 

of nucleus and cell substance. Careful examination of the nuclear structure 
with its wide-open meshwork, should, however, prevent the mistake of classi- 
fying this cell as a large lymphocyte. 

The clinical significance of the megaloblast is more or less in doubt. As 
such cells are foreign to the blood of an adult and as they are not present beyond 
15 per cent, of the total nucleated reds in the marrow, according to Emerson, 
it must be assumed that their presence in considerable number is indicative 
of a reversion to an embryonal type of blood formation, or at least denotes 
an arrested development of normal cells and in consequence an increased 
production of these abnormal types. Megaloblasts are, therefore, evidences 
of degeneration of blood-forming organs, while normoblasts are significant 
of regeneration of blood. A few of these cells, along with a larger number of 
normoblasts, has no special significance in cases of severe anemia, being 
found in small numbers in any variety of anemia. If, however, the majority of 
the nucleated red cells be megaloblasts, especially if gigantoblasts be present 
and unequal mitoses be observed, a diagnosis of primary pernicious anemia seems 
justified. It may be considered that the number of erythroblasts has no special 
significance as regards the severity of a particular case, but that it indicates 
merely the effort on the part of the bone-marrow to overcome the effects of 
blood destruction. The appearance of megaloblasts may, therefore, be regarded 
as an evidence of incomplete formation of the younger elements and, not 
necessarily, as an unfavorable sign. In the anemia following infection with 
bothriocephalus latus, the specific toxins produce a megaloblastic degeneration 
of the bone-marrow, so that the blood picture may assume the characteristics 
of a severe pernicious anemia. Ehrlich and Lindenthal have reported a case 
of nitrobenzol poisoning, in the later stages of which the megaloblasts out-num- 
bered the erythroblasts. Thayer 1 is the first to report the finding of ameboid 
movements in a megaloblast in a case of Addisonian anemia. 
(£>). Number of Red Cells. 

The normal number of red cells is generally regarded as 5,000,000 per cmm. 
in the blood of the adult male, while the normal value for the female is 4,500,000. 
These figures are purely arbitrary, but they serve as an approximate basis upon 
which one may form his opinion as to the probable normality of a specific speci- 
men of blood. We frequently observe marked variations in the red count under 
the influence of both physiologic and pathologic conditions. These variations 
may be due to an actual increase or decrease in the number of cells or to a change 
in the volume of the plasma as discussed under Total Volume of Blood on page 
388. It is not uncommon to find much higher counts than these normal ones in 
healthy individuals, especially in those living the " simple life," so that any special 
count must be a law unto itself and must be considered normal or abnormal 
only after considering all the factors which may influence the number of cells. 

Physiologic Variations. 
(1). Sex. 

The variations due to sex are not so marked as those from other causes. 
While the adult woman almost invariably shows a lower blood count than does 

1 Arch. Int. Med., 1911, VII, 223; Ibid., 1911, VIII, 581. 



THE BLOOD. 493 

the adult man, yet we occasionally find the girl showing, before puberty, a 
somewhat higher count than the boy of corresponding age, and also the woman, 
after the menopause, showing somewhat higher values than does her brother of 
similar years. It can hardly be doubted that menstruation, as well as pregnancy 
and lactation, have some influence in lowering a count in woman at certain 
periods of life, yet these conditions are simply transient and would not seem to 
have as much bearing on the question as does the somewhat more hydremic 
state of the normal plasma of woman. 
(2). Age. 

The number of the red cells varies more or less with the age of the subject 
examined. The highest values are usually observed at birth, when the count 
may run as high as 7,500,000, as in a case observsd by the writer, the hemoglobin 
being practically always over 100 per cent, at this time; but, as a rule, a lower 
count is noted, averaging about 6,000,000. These high figures are due, to 
some extent, to the concentration of the plasma at birth through the loss of 
body fluid before a compensatory intake. The count gradually falls during the 
first few days and becomes fairly constant about the tenth day, when it is rare 
to find nucleated red cells. The number of cells is then stated to be somewhat 
reduced until the age of puberty, when a gradual increase occurs until about 
40 years of age, after which a slight decrease may be observed in man. These 
variations, observed at different ages, are to be regarded as slight and influenced, 
to some extent, by the many modifications of the plasma as the result of growth 
and development as well as to the gradual decrease of functional activity after 
the middle periods of life. 
(3). Altitude. 

An increase in the number of cells has been observed under the influence 
of a higher altitude. The count has heen shown to increase at the rate of 
approximately 50,000 cells per 1,000 feet of ascent and to diminish, within 
36 hours, at a corresponding rate, this increase or decrease being more marked 
the more sudden the ascent or descent. Just what factors are at work in causing 
these changes is undecided. 1 The rise is too rapid to be entirely accounted for 
by new formation of blood-cells under the influence of diminished oxygen tension, 
and the fall is not accompanied by signs of destruction of the reds. Weinzirl 
considers the increased count of high altitudes due to the lowered temperature 
at these elevations. This factor would seem to have some influence, as counts 
are frequently observed in which a variation is noted at places showing the 
same elevation. Change of residence from warm to a cold or from cold to a 
warm climate may lead to an increase or decrease, 2 as the case may be, in the 
number of cells varying from 500,000 to 2,500,000. 
(4). Nutrition. 

The general nutritive condition of the subject has more or less influence 

1 See Cohnheim, Kreglinger, Tobler, and Weber, Ztschr. f. physiol. Chem., 1912, LXXVIII, 
62; Schneider and Havens, Am. Jour. Physiol., 1915, XXXVI, 380. 

2 Chamberlain has, however, shown (Philippine Jour. Sc, Sec. B., 1911, VI, 467) that the 
average number of red cells in the tropics is little different from that in temperate zones. 
See Laquer, Deutsch. Arch. f. klin. Med., 1913, CX, 189; Cohnheim and Weber, Ibid., 225; 
and Lope, Semana Med., 1913, XX, 1077. 



494 



DIAGNOSTIC METHODS. 



upon the number of red cells per cmm. This statement must not be interpreted 
to mean that the obese person shows a higher blood count than does his sparer 
brother. It is well known that obesity is an indication of poor assimilation of 
food; hence we should expect to find, as we really do, that the muscular person 
has a somewhat higher count than the obese subject and well-developed robust 
individuals a larger value than do the poorly nourished patients. Diet has, of 
course, much to do with the general nutrition, so we find the meat eaters averag- 
ing somewhat higher in their percentage of red cells than do vegetarians. 
Immediately following a hearty meal we may observe a temporary decrease in 
the number of red cells, but this soon returns to normal, owing to the rapid 
adjustment of the water content of the blood. 1 

(5). Exercise. 
Active muscular exercise produces a transient increase in the red count 
due both to the increased blood-pressure and to the concentration of the blood 
through the loss of water by perspiration. Physical exercise, taken to the 
point of producing fatigue, may produce a marked diminution in the number 
of red cells, due probably to the fact that regeneration of new cells cannot keep 
up with the destruction of the old cells. Passive exercise, in the form of 
massage, has a transient influence in producing an increase in the number 
of the red cells, owing to its increasing the general circulatory tone. 2 

(6). Baths. 
Careful investigation of the influences of both cold and hot baths has shown 
that an increase in the number of reds occurs under the action of these two 
conditions. The increase as a result of a cold bath may be as great as 2,000,000 
cells, due to capillary stasis as a result of vasomotor constriction. Likewise, 
a hot bath will increase the number of cells by causing dilatation of the pe- 
ripheral vessels and a consequent increase in the amount of blood at the point 
from which the specimen is taken. When marked perspiration follows either 
a hot or cold bath the blood becomes concentrated and, as a result, an increase 
in the number of red cells will obtain. 

(7). Therapeutic Measures. 

Any drugs which cause rapid loss of fluid from the body, as for instance 
emetics, purgatives, diuretics, and diaphoretics, will cause concentration of 
the blood and hence a coincident increase in the number of cells, providing 
the change is sufficiently rapid and the blood examination is made before 
compensation occurs. Among the drugs which increase the number of red 
cells we find iron and arsenic, both of which are particularly valuable in anemic 
conditions, the former in chlorosis and the latter in pernicious anemia. The 
compounds of mercury and of lead, on the other hand, have a destructive 
action upon the red cells, so that we find these drugs causing a diminution in 
the number of cells. 



239- 



1 See Hopmann, Arch. f. Verdauungskr., 1913, XIX, 456. 

2 See Hawk, Am. Jour. Physiol., 1904, X, 384; Schneider and Havens, Ibid., 1915, XXXVT, 



THE BLOOD. 495 

Pathologic Variations. 
(1). Oligocythemia. 

This is a condition characterized by a diminution in the number of red 
cells. It is usually associated with a decrease in the percentage of hemoglobin 
and with a slight reduction in the total volume of blood, although this latter 
factor is not invariably present. This condition is found, to a more or less 
degree, in all forms of anemia and may be temporary or permanent. The 
extent of the decrease in the number of red cells varies from 500,000 cells to 
a reduction of 4,000,000. This diminution in the number of cells is usually 
an indication of the severity of the anemia, the most marked decrease being 
observed in the pernicious types, Osier reporting probably the lowest count 
recorded, namely, 100,000 red cells. 

While this oligocythemia is usually associated with oligochromemia, yet 
we find in chlorosis that the diminution of red cells is not as marked as is the 
reduction in the amount of hemoglobin, the color index in this condition being 
usually low. In some cases of chlorosis, however, the oligochromemia may 
keep pace with the oligocythemia. In pernicious anemia, on the other hand, 
we have a marked reduction in the number of cells and coincidently a large de- 
crease in the amount of hemoglobin, the result being that we have a very high 
color index as the usual sign of this condition. Cases showing the loss of a 
large amount of blood, as the result of hemorrhage have naturally an oligocy- 
themia, but the percentage of hemoglobin may not necessarily be reduced to 
any great extent. This gives rise to the condition of secondary anemia, in which 
the color index may be high. In these cases of hemorrhage a sudden reduction 
to the point of 1,000,000 or less cells is usually followed by a fatal result, although 
even here recovery has been made possible by rapid infusion of salt solution 
and compensatory activity of the blood-forming organs. In leukemia we find 
that the red cells are not usually diminished to a very great extent, the oligo- 
cythemia being generally more marked in the lymphatic than in the myeloid 
variety. Occasionally, however, we do find very low counts, in both varieties 
of leukemia. These same statements apply to splenic anemia, although the 
count here very rarely reaches a lower point than 2,500,000 cells. 

A large number of conditions, aside from direct blood diseases, cause a 
diminution in the number of these red cells. Thus we find that the toxins 
of certain of the specific fevers, such as typhoid and pneumonia, may cause 
a marked anemia, but this is not necessarily the rule. Acute infection with 
pus organisms is frequently observed to cause an extreme and rapidly progress- 
ive anemia, the destruction of the red cells being in some cases very extensive. 
In malignant disease we usually find, especially if cachexia is present, a very 
extensive anemia which may lead to a diagnosis of the pernicious type. It is 
to be remembered, however, that a secondary anemia may assume all of the 
characteristics of the primary pernicious type. 
(2). Polycythemia. 

This is a condition characterized by an increase in the number of red 
cells and is sometimes called polyglobulia. 1 Whether this increase is actual 

1 Also erythremia. 



496 DIAGNOSTIC METHODS. 

and permanent or whether it be simply apparent, due to concentration of 
the blood or to unequal distribution of blood in the peripheral vessels, is 
still an unsettled question. It is true that many physiological causes previously 
enumerated do bring on a polycythemia, so that these should be remembered 
whenever a blood count is to be made. From the pathologic standpoint 
we find an increase in the number of red cells occasionally following active 
blood regeneration after hemorrhage. Likewise, we observe an increase in the 
number of cells in phosphorus poisoning, acute yellow atrophy of the liver, and 
in certain cases of general hepatic insufficiency. Why such an increase of cells 
occurs in these conditions is very hard to say. It has been found that the solids 
of the plasma are not increased, so that we are not warranted in assuming a 
concentration of the fluid elements. A polycythemia of uncertain origin is 
also seen in cases of poisoning with carbon monoxid and illuminating gas. 1 

In a condition called by Osier autotoxic enterogenous cyanosis, which is 
characterized by a marked increase in the number of red cells and also by 
enlargement of the spleen, we have reason to assume that a direct real increase 
in the number of red cells occurs, but the pathogenesis of the condition is uncer- 
tain. It has been traced to improper aeration of the blood and occurs 
also in congenital heart disease, mitral lesions in the adult, and in pneumonia 
and acute miliary tuberculosis. While this high blood count must be definitely 
admitted, its importance from a clinical standpoint is still unsettled. It is 
uncertain whether the rapid increase in the number of reds is due to an attempt 
on the part of the blood-forming organs to overcome the influences of the toxic 
substances upon proper oxidation in the system; yet it is certain that relief very 
frequently follows such an increase in the number of red cells, although in 
Osier's disease or, as it is called by some, Vaquez's disease, death frequently 
follows without any relief from the condition. 2 

(E). Staining Properties of the Red Cells. 

The normal red cell, like all living cells, is incapable of being stained with 
anilin dyes; that is, it is achromatophilic. Previous to staining, fixation of the 
cell must take place, the protoplasm being killed in this process. The normal 
fixed red cell has a marked affinity for various dyes of the acid type, such as 
eosin, orange-G, acid fuchsin, and indulin, and is therefore called acidophilic or 
oxyphilic. This cell normally takes up but one color from a mixture of dyes, 
and is called, therefore, monochromatophilic. 

Polychromatophilia . 

Under various pathologic conditions we find red cells which show a tendency 
to take up the basic stains, and for this reason are called basophilic or poly- 
chromatophilic cells. The tint of such abnormal red cells varies from that of a 
light indefinite shade of the basic stain to a dark distinct tone. Just what 
factors are at the bottom of this change, which is called polychromatophilia 

1 See Karasek, Trans. Chic. Pathol. Soc, 19 11, VIII, 173. 

2 See White, Lancet, 1912, XCII, 7; Lucas, Arch. Int. Med., 1912, X, 597; Cahn, Inaug. 
Dissert, Berlin, 1912; Chauffard and Troisier, Presse med., 1913, XXI, 653; Friedman, Med. 
Record, 1913, LXXTV, 701; and Moewes., Deutsch. Arch. f. klin. Med., CXI, 281; Hertz 
and Ehrlich, Deutsch. Arch. f. klin. Med., 1914, CXVI, 43; Mosse, Ztschr. f.klin. Med., 1914, 
LXXLX, 431; Lamson, Jour. Pharmacol, and Exper. Therap., 191 5, VII, 169. 



THE BLOOD. 497 

or polychromasia, is difficult to say. The normal acidophilic tendencies of the 
cell are due to the presence of hemoglobin and, in consequence, the normal 
staining properties will depend upon the relative richness of the cell in this 
pigment. As hemoglobin is always acidophilic, we cannot assume that the 
polychromatophilic properties of the abnormal red cells are due to variations 
in the hemoglobin, but must content ourselves with the belief that these changes 
have something to do with the protoplasm of the cell. 

This condition, which has been termed by Ehrlich anemic degeneration, 
is characterized by a diffuse basic staining property of the red cells. These 
basophilic cells are somewhat larger than the normal ones, show less bicon- 
cavity, and often are abnormal in shape. The megaloblasts are practically 
always polychromatophilic and the normal erythroblasts of the bone-marrow 
usually show this degeneration. This fact would seem to point to the probability 
of this condition being a sign of regeneration of the blood as the erythroblasts 
are more prone to be polychromatophilic the younger they are. 

Two distinct forms of polychromasia have been found. The first of 
these, known as the polychromatophilic degeneration of Gabrits chew sky, is the 
diffuse basophilic staining of the cells which is found in various forms of 
anemia and extensively in the cells of the normal bone-marrow. The second 
of these, the polychromasia of Maragliano, is shown in severe anemias and 
in toxemias and appears as a more punctate basophilia, being closely related 
to the basophilic degeneration of Grawitz, which will be discussed later. While 
the polychromatophilia of the diffuse type is considered by Ehrlich as evidence 
of a degenerative process, that is as a coagulation necrosis of the discoplasm 
in consequence of which this takes up the albuminous principles of the plasma 
while it loses its power of retaining hemoglobin, yet there are evidences showing 
that this may be a regenerative process. 1 Polychromatophilia is often seen in 
cells which are undergoing degeneration, especially in myelogenous leukemia, 
pernicious anemia, severe secondary anemia, especially those following malig- 
nant disease, eruptive fevers, malaria, and after various poisons, such as that of 
snake venom. 

The forms of partial polychromatophilia, which have been described 
under various names, such as vacuolization, pseudonucleation, globular 
decolorization, and more commonly Maragliano's endoglobular degeneration, 
are seen in normal blood in from 30 to 70 minutes after the specimen is made. 
They are usually found in the center of the cell, but may be near the periphery. 
There may be several such areas in a single cell, but the more common form 
is the single area of degeneration, which is usually round, may be elliptical, 
and may resemble a vacuole. These are shown quite distinctly in the unstained 
specimen and in this condition very frequently are seen in rapid motion. 
This motion is not of the true ameboid type, but is due more to the gradually 
progressing coagulation and consequent constriction of the protoplasm. These 
areas, whether in the fresh or stained specimens, may be mistaken for malarial 
parasites, so that the worker should be on his guard lest he make a wrong diag- 
nosis without sufficient evidence. They differ in size from the parasites, and 

1 See Pfuhl, Ztschr. f. klin. Med., 1913, LXXYIII, 102. 
32 



498 DIAGNOSTIC METHODS. 

on focusing the specimen are characterized, usually, by a change from a smaller 
to a larger form, which variation is not evident with the malarial organism. 
The longer one searches in the unstained specimen for malarial organisms the 
more apt a mistake in diagnosis is to be made, as these " Maraglianos " develop 
quite rapidly. In the stained specimen these areas of coagulation necrosis will 
show a basophilic staining quality, while the chromatin element of the malarial 
organism will differentiate this body from the more usual degenerative area. 
(F). Degenerations. 
While the preceding conditions of polychromatophilia are to a certain 
extent true degenerations, yet they are more properly grouped under the heading 
of atypical staining reactions of the red cell. 

Basophilic Degeneration of the Red Cells. 

This condition is distinct from the polychromatophilia above described 
and is known as the punctate basophilia of Grawitz. It is characterized by 
the appearance, in the body of the red cell, of granules of varying size, which 
stain with the basic dyes. The cell may be dotted throughout with these gran- 
ules or may show this degeneration only in parts. These granules are not 
observed in the fresh unstained specimen and are not increased, as are the 
Maragliano areas, by allowing the blood to stand. These cells are observed 
in pernicious anemias, leukemia, in the toxemia of malignant states and espe- 
cially in cases of lead-poisoning. 

The size of the granules may vary from small dots to large granules, 
showing a diameter of one or more microns. Their origin is much in doubt, 
but they are generally considered to be areas of coagulation necrosis associated 
with either degeneration or regeneration of the red cells. They are quite 
distinct from the granular basic "stippling" observed in malarial conditions, 
but are probably evidences of direct toxic effects upon the protoplasm of the 
red cells. Their greatest clinical importance is probably in lead poisoning, 
where they may be the only signs of abnormality in the blood picture. They 
may vary in number from day to day, showing in some cases five or six in one 
microscopic field, but, as a rule, they are present in much fewer numbers, as 
one observes them only after examining several visual fields. As a rule, they 
appear very early in cases of lead-poisoning, in one case observed by the 
writer within three days, and they may be present in the blood of a lead worker 
for several years after his exposure to the effects of the lead. They are usually 
the first sign of anemic change and usually persist longer than do the other 
abnormalities of the blood. 1 

Ring Bodies. 

Occasionally one observes in the red cells curious ring-like bodies which 
are in shape very much like the hyaline malarial ring with a circular refractive 
center. They change their shape in a peculiar way, much resembling the 
undulatory movements of the hyaline body. They do not increase in number 
or grow larger on standing as do the Maraglianos and they are observed in 

1 See Petry, Biochem. Ztschr., 1912, XXXVIII, 92; Anders, Jour. Am. Med. Assn., 1914, 
LXII, 1164; Linenthal, Ibid., 1796; Schnitter, Deutsch. Arch. f. klin. Med., 1915, CXVII, 127. 



PLATE XIX. 



O 











Xathar ir\e MUl 



Ring Bodies in Red Cells. (After Schleip.) 



THE BLOOD. 499 

a large number of conditions, such as measles, pernicious anemia, and severe 
secondary anemia. Two types of these ring bodies must be distinguished, 
the first is usually more peripherally situated, occasionally has a definite 
crescentic shape or has an appearance much resembling that of a Maragliano, 
and is found especially in cases of measles. This finding has lead to the assump- 
tion that these bodies were the organisms causing measles. The second form 
has been described by Cabot 1 and more recently by Schleip 2 in cases of perni- 
cious anemia, leukemia, and secondary anemias. These latter bodies are 
larger than the former, more distinct, irregular in shape, frequently forming 
figure-eight bodies and are usually stained bright red with Wright's stain, but 
may occasionally show a blue shading. Herrick 3 has observed thin, elongated, 
sickle-shaped red cells, some of them nucleated, in a case of severe anemia. 

Various other forms of degeneration of the red cells occur, as for instance 
the appearance of rod-like areas resembling bacilli, 4 which may keep up a con^ 
stant vibratory motion carrying them through the entire substance of the cell. 
This finding should not confuse one in making a diagnosis of the presence of 
bacteria. Another form of degeneration, known as Ehrlich's hemoglobinemic 
degeneration, has the appearance of a small dark cell lying upon a larger paler 
one. These are probably areas of condensed protoplasm with the hemoglobin 
distinctly separated from the stroma. Such cells appear, occasionally, in 
certain types of malaria and are shown as corpuscles in which the hemoglobin 
is apparently condensed around the parasite. This degeneration is occasionally 
seen in nucleated red cells and may give the appearance of a microblast lying 
upon a macrocyte. It is best seen in cases of pernicious anemia arid may 
explain some of the "acidophilic granules" of the red cells which have been 
described (Emerson). 

(G). Isotonicity and Resistance of the Red Cells. 

Normally, the relations of the cellular to the fluid portions of the blood are 
such that the hemoglobin and other constituents of the red cells are held intact 
within the limiting membrane of the cell. Any change in the osmotic pressure 
of the plasma is certain to be manifested by variations both in size and in the 
composition of the cellular elements. We find, therefore, as previously discussed 
under the heading of Osmotic Pressure, that the inorganic constituents of the 
plasma are the factors upon which the volume of the red cell depends to a 
large, extent. 

By an isotonic solution, as applied to the blood, we mean a solution of 
such a strength as to preserve the corpuscles and to prevent passage of water, 
salts, and organic bodies from the plasma into the corpuscle or from the corpus- 
cle into the plasma. In other words, an isotonic solution is one whose osmotic 
pressure is equal to that of the contents of the red cell. While the limiting 
structure of the red cell is indefinitely known, it seems to have certain properties 

1 Jour. Med. Research, 1903, IX, 15. 

2 Deutsch. Archiv. f . klin. Med., 1907, XCI, 449. 

3 Arch. Int. Med., 1910, VI, 175. See, also, Bishop, Arch. Int. Med., 1914, XIV, 388; 
Cook and Meyer, Ibid., 1915, XVI, 644; Pisani, Folia Hsemat., 1915, XlXf(i), 119. 

4 See Roth, Ztschr. f. klin. Med., 1912, LXXVI, 23. 



500 DIAGNOSTIC METHODS. 

which would lead us to assume that it is a semipermeable membrane, that is 
one which will permit the passage of organic compounds or of salts or their 
ions in both directions. This apparent selective activity is dependent both 
upon the laws of osmosis and diffusion through a semipermeable membrane 
and upon the basic ideas of Ehrlich's side-chain theory. While other figures have 
been used at various times to represent the strength of a solution isotonic with 
the red blood-corpuscle, the one which seems more nearly to duplicate the 
actual condition is a 0.9 per cent, solution of sodium chlorid. This is or should 
be the normal salt solution which is used in physiological work and in transfu- 
sion. Loeb has shown that a properly balanced solution containing the chlorids 
of sodium, potassium, and calcium more nearly represents a proper "physio- 
logical salt solution" than does the simple 0.9 per cent, sodium chlorid solution. 
Such an isotonic solution will preserve the corpuscles, will prevent the passage 
of hemoglobin from the cells into the plasma, and will not permit of shrinkage 
or swelling of the cells. 1 Solutions which are stronger than the one above 
mentioned are called hypertonic solutions, while those of a lower concentra- 
tion are termed hypotonic solutions. In the first of these shrinkage of the 
cells will occur, while in the latter phenomena of swelling will be observed. 
The erythrocyte normally shows a certain amount of resistance to variations 
in the osmotic pressure of the blood and to the hemolyzing effects of various 
substances. It is true that the osmotic pressure of the plasma may be raised 
by the introduction of certain substances into the blood without causing 
phenomena of shrinking, swelling, or hemolysis to occur, yet we must not assume 
that the cell is not influenced by such changes in osmotic pressure. Simple 
variations in osmotic pressure come more nearly to the foreground in the 
explanation of these changes, especially of those occurring in most disease 
processes of a chronic type, while in the more acute infectious types associated 
with toxemia the biologic theory must be invoked. As yet little of value has 
been forthcoming from the study of the resistance of the blood-cells to variation 
in osmotic pressure, as the scope of the examination has not been sufficiently 
widened by application of accurate experimental methods. The writer is 
forced to say, from the results of his own experiments, as well as those of 
others with the more usual method of Hamburger, that no reliable data are 
at hand. It is highly probable that the little understood biologic influences 
are clinically much more important than are those due to variations in osmotic 
pressure, as this latter process cannot explain the hemolytic results of the toxins 
or of certain definite chemical compounds which bring about these results in 
concentrations which, have little influence upon the tonicity of the blood. 2 

(H). Variations of the Red Cells in Childhood and in Old Age. 

As previously stated, the average number of red cells is much higher than 
the normal figure at birth, gradually decreasing until the age of puberty when 

1 Ringer's solution is frequently used in this work. Its composition is: sodium chlorid, 9 
grams; calcium chlorid, 0.24 gram; potassium chlorid, 0.42 gram; sodium bicarbonate, 0.2 
gram; water, 1,000 c.c. 

2 See Pepper and Peet, Arch. Int. Med., 19 13, XII, 81; also, Gaisbock, Deutsch. Arch. f. 
klin. Med., 1913, CX, 413; Holler, Ztschr. f. klin. Med., 1914, LXXXI, 129; Hober, Deutsch. 
med. Wchnschr., 1915, XLI, 273; Roccavilla, Rif. Med., 1915, XXXI, 813 and 873. 



THE BLOOD 50I 

a gradual increase occurs again until middle life, after which a steady decrease 
is observed. It is to be expected that variations in the color tones (hemoglobin 
content) of these cells will vary normally with the age of the subject, being very 
high in the early days of life, gradually diminishing as puberty approaches, in- 
creasing until the middle periods of life, and steadily declining as the age 
increases. Aside from the number of cells, the blood in childhood shows little 
variation in the characteristics of the red cells, but the variations in the plasma 
are more or less marked, as the synthetic and retrograde products of tissue 
metabolism are much more evident in the period of development than during 
the middle periods of life. 

As the subject becomes older the blood is laden with products of retrograde 
tissue change and the blood-forming organs become less active. It must 
be remembered that the various organs of the body contribute certain factors 
to the blood and may, therefore, all be considered as blood-forming organs in 
a definite sense. The ductless glands, in particular, pour into the blood 
certain substances which markedly influence the proper correlation of these 
various organs. Just what these substances are is not well established, so that 
the introduction of the word "hormone" by Starling does not at present have 
a specific meaning. As old age approaches variations become evident in the 
plasma, and through such influences changes in the morphology of the red 
cells are more or less frequent. Besides these plasma changes, gradual atrophic 
degeneration of the blood-forming organs takes place, so that we may find red 
cells showing the various types of anemic degeneration and may even see 
nucleated red cells as an evidence of the attempt on the part of the bone-marrow 
to overcome the gradual destruction of cells. The more notable variations 
in the red cells are observed in their size, owing to the hydremic state which is 
almost always characteristic of the plasma in advanced periods of life, but 
associated diseased conditions will, of course, give their characteristic changes. 

(/). Functions of the Red Blood-cells. 

The only well-recognized function of these cells is their oxygen- carrying 
power, which is dependent upon their hemoglobin content. Other oxygen 
catalysts, such as oxidase, peroxidase, and catalase, are found more largely in 
the leucocytes and play a significant role in the function of the whole blood. 
These will be discussed later. As is well known, the fluid portions of the blood 
are much more influenced by the various organs of the body than are the 
cellular elements, although the red cells will naturally be affected by variations 
in the hematopoietic organs, such as the bone-marrow, liver, and spleen. Our 
knowledge regarding the variation in the plasma is so uncertain that we have 
been led to interpret the changes in the histological appearance of the blood as 
significant of certain general pathological conditions. It is true that in some 
cases we do have direct disease of the blood-forming organs and, in consequence, 
variations in the cellular structures are of diagnostic importance, yet in the 
large majority of cases the preliminary changes must be resident in the plasma, 
those in the cellular structure being simply incidental. It may be, therefore, 
necessary to assume a functional activity on the part of the red cells to overcome 



502 



DIAGNOSTIC METHODS. 



biologic changes in the plasma and if so we must attempt to discover just what 
plasma changes we are to regard as significant of the various conditions shown 
by the red cells in each true blood disease. 

(6). Leucocytes. 
(A). Appearance. 
In the fresh specimen the leucocytes appear as colorless, highly refractive 
bodies, somewhat larger than the red cells, and showing a definite nucleus. 
These white cells show distinct ameboid movement in the fresh specimen 
by virtue of which they are able to surround a foreign body and enclose it 
within their own protoplasm. This is the well-known property of phagocytosis 
which is such an important factor in Metschnikoff's theory of immunity. 
Tn contradistinction to the red cells, these white cells show many variations 
both in size and in shape. As the relations between the nucleus and the cellular 
protoplasm is quite distinct in many of these forms, some writers have been 
led to classify the white cells according to the peculiarities of the nucleus. 
Other writers classify them into granular and nongranular forms, as many of 
these cells show distinct granulations which are more clearly differentiated in 
the stained specimens. As more or less confusion exists regarding these different 
variations, it seems more rational at present still to classify these white cells 
with Ehrlich, who combines both the nuclear and granular characteristics in 
his classification. 1 

(B). Leucocytes in Normal Blood. 
(i). Lymphocytes. 

The cells of this class which occur normally in the blood are of two kinds, 
(a) the small cell, about 5 to 8 microns in diameter and (b) large cells showing 
a diameter of from 8 to 10 microns. Both of these cells have a very large 
nucleus which is usually centrally located but may have an eccentric position. 
It may be compact or coarsely reticulated and is not always as strongly stained 
as is the surrounding cytoplasm. Nor is it as deeply stained as is the nucleus 
of the normoblast, although it is very rich in chromatin. The nucleus of the 
larger cell may show very irregular staining properties. The nuclei of both of 
these cells is usually circular in outline, but may show an oval or a kidney- 
shaped structure, in which at times may be seen a distinct nucleolus. The 
protoplasm of these cells is usually in the form of a narrow rim surrounding 
the nucleus and showing a strongly basophilic homogeneous cytoplasm. The 
older cells may at times show a difficult staining property, may even be acido- 
philic, and occasionally exhibit a net-like structure in which are observed a 
few granules scattered throughout the protoplasm of the cell. This granulation, 
which occurs in about one- third of the cells seems to have little clinical signifi- 
cance, except as a .probable sign of age of the cell, this granular stage being 
regarded as the end-point of their development. These granules are probably 
not representatives of true granulation, but seem to be more referable to nodal 
points of the reticular structure of the protoplasm. 

1 See Pappenheim, Die Ergebnisse der inn. Med. u. d. Kinderheilkde., 1913, VIII, 183; 
also, Hoxie, Interstate Med. Jour., 1913, XX, 1049. 



DESCRIPTION OF PLATE XX. 



( Triacid Slain.) 

1, 2, 3, 4. Small Lymphocytes. 

Contrast the faintly colored protoplasm of these cells in the triple stained specimen 
with their intensely basic protoplasm in the film stained with eosin and methylene- 
blue, 17 and 18. The cell body of 1 is invisible. Note the kidney-shaped nucleus in 4. 

5, 6. Large Lymphocytes. 

With this stain the nucleus reacts more strongly than the protoplasm: with eosin 
and methylene-blue (19, 20), on the contrary, the protoplasm is so deeply stained 
that the nucleus appears pale by contrast. This peculiarity is also observed in the 
smaller forms of lymphocytes. 

7, 8. Transitional Forms. 

Note the moderately basic and indented nucleus, and the almost hyaline non- 
granular protoplasm. Compare 8 with the myelocyte, ;. Plate IV, these cells dif- 
fering chiefly in that the myelocyte contains neutrophile granules. 

9,10,11. Polynuclear Neutrophiles. 

These cells are characterized by a polymorphous or polynuclear nucleus, sur- 
rounded by a cell body filled with fine neutrophile granules. In 11 the nuclear 
structure is obviously separated into four parts; in 9 it is moderately, and in 10 
markedly, polymorphous. 

12, 13. Eosinophiles. 

The nuclei are not unlike those of the polynuclear neutrophile, except that they are 
somewhat less convoluted, and poorer in chromatin, staining less intensely. The 
protoplasm is filled with coarse eosinophile granules, the characteristics of which 
are clearly illustrated by 13, a " fractured " eosinophile. 

14. Eosinophilic Myelocyte. 

Compare with 15. 

15, 16. Myelocytes. {Neutrophilic.) 

These cells are morphologically similar to 14, except that they contain neutrophile 
instead of eosinophile granules. Note that the granules of the myelocyte are 
identical with those of the polynuclear neutrophile. A dwarf form of myelocyte is 
represented by 16. 

{Eosin and Methylene-blue.) 

17, 18. Small Lymphocytes. 

Note the narrow rim of pseudo-granular basic protoplasm surrounding the nucleus 

and the pale appearance of the latter. 
19,20. Large Lymphocytes. 

Budding of the basic zone of protoplasm is represented by 20, Both of these cells 

belong to the same type as 5 and 6. 
21, 22. Large Mononuclear Leucocytes. 

Compared with 19 and 20, these cells have a decidedly less basic protoplasm, but a 

somewhat more basic nucleus. In the triple stained film these differences cannot 

be detected, so that they must be classed as large lymphocytes. 
23. Transitional Form. 

The distinction between this cell and 24 is not marked; the nucleus of the latter 

simply being somewhat more basic and convoluted. 
24, 25, 26, 27. Polynuclear Neutrophiles. 

With this stain these cells show a feebly acid protoplasm, and lack granules. Note 

that the more twisted the nucleus the deeper it is stained. Compare with 9, 10, 

and 11. 
28, 29. Eosinophiles. 

Compare with 12 and 13. 

30. Eosinophilic Myelocyte. 

Compare with 14. 

31. Basophile. {Finely granular.) 

This cell is characterized by the presence of exceedingly fine 5-granules, staining 
t)\&pure color of the basic dye. The nucleus is markedly convoluted and deficient 
in chromatin. The cell here shown was found in normal blood. 

32. 33, 34, 35, 36. Mast Cells. 

The granules take a modified basic color, as shown by their royal-purple tint in this 
illustration. Note their unusually large size and ovoid shade in 35, their peculiar 
distribution in 35 and 36, and their irregularity in size in 32 and 36' With the triacid 
mixture these granules, as well as those of the finely granular basophile, 31, remain 
unstained, showing as dull-white stippled areas in the cell body. The nuclear chro- 
matin of the mast cell is so delicate and so freely stained that it is barely visible. 
These cells were found in the blood of a case of spleno-medullary leukemia. 



PLATE XX. 



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The Leucocytes. 

(i-i6, Triacid Stain ; 17-36, Eosin and Methylene-blue.) 

(E. F. Faber,/<?c.) 

(From 73a Costa's "Clinical Hematology.") 



THE BLOOD. 503 

These cells usually constitute from 20 to 25 per cent, of the leucocytes v 
their absolute numbers being from 1,200 to 2,000 per cmm., but the relative 
proportion of the small and large form being unsettled. The larger types of 
these cells are more closely associated with increased functional activity of the 
lymphoid tissues and should probably be considered the older cell of the 
two. According to Pappenheim, the large lymphocyte represents the mother cell 
from which all other leucocytes as well as erythrocytes are indirectly developed 
as a result of heteroplastic differentiation. Variations in technic of pre- 
paring the slides will often lead to differences in size of these cells, the thinner 
preparations showing these cells as larger forms, while the thicker preparations 
present the smaller type of these cells. 

These cells are beautifully colored by the hematoxylin-eosin stain and 
by the various modifications of the Romanowsky stain, although the boundary 
between the nucleus and the protoplasm is not always clearly outlined by 
these latter stains. The granules, which are occasionally found in these cells 
and which are in size between the <*■ and the e granules of Ehrlich, show most 
clearly in preparations stained by the Giemsa stain and appear distinctly 
azurophilic. These granules do not appear at all with the triple stain. 

These cells are frequently increased and frequently diminished, the 
former condition being spoken of as lymphocytosis, the latter as lymphopenia. 
A relative increase of the number of these lymphocytes is regarded as more or 
less characteristic of typhoid fever, especially when associated with a diminution 
in the total number of white cells. In lymphatic leukemia these cells are 
present in large numbers and are indicative of marked disturbance in the 
lymphatic structures. 

(2). Large Mononuclear Leucocytes. 

These cells, which are supposed to be derived from the spleen and are 
called, therefore, splenocytes, are as a rule two or three times as large as a 
red cell (12 to 15 microns), and show a nucleus which may be large and round, 
but is more frequently oval in shape or may be indented, forming the so-called 
kidney type of nucleus. It is to be remembered that we may have both large 
and small types of this large mononuclear leucocyte, the smaller forms being 
distinguished from the lymphocyte by the relation of the protoplasm to the 
nucleus. These nuclei are usually eccentric in position and are not always 
sharply outlined, they are poor in chromatin, but are strongly basophilic, 
although less so than are the nuclei of the lymphocytes. The protoplasm 
of these cells is very abundant in relation to the size of the nucleus and is very 
clear, hyaline, and nongranular in appearance. This protoplasm is much less 
basophilic than is the nucleus and shows a very fine reticulum with nodal 
thickenings which are somewhat more strongly basophilic and may give the 
appearance of granulation. 1 These cells should not, however, be regarded 
as granular cells. 

Occasionally we find cells, derived from the above, which have been called 

" * Bergel (Deutsch. Arch. f. klin. Med., 1912, CVT, 47) has shown that these cells 
play a large role in the hemolysis and lipolysis noted after injection of cells and lipoids. 



504 DIAGNOSTIC METHODS. 

by Ehrlich "transition forms," which are the largest of all the white cells. 
The nucleus is pale, often deeply notched giving it usually the appearance of 
a saddle-bag, and shows the characteristics of the large mononuclear type, 
being distinguished from the polymorphous nucleus of the neutrophile cells 
by its greater thickness and by its diminished intensity of staining. The 
extensive granulation of the polymorphonuclear leucocyte should make any 
mistake in diagnosis impossible. The protoplasm of these transitional cells 
is very abundant and shows faint basophilic properties and may even have 
a few fine granules, which are neutrophilic in character, in the neighborhood 
of the nucleus. These two forms of cell constitute between 3 and 5 per cent. 
of the leucocytes, their absolute number varying between 200 and 400 per cmm. 
Pappenheim considers that these large mononuclear cells develop directly 
from the large lymphocytes and then pass into the transition forms, which are 
the final developmental types. 

(3). Polymorphonuclear Cells. 
(a). Polymorphonuclear Neutrophiles. 

These cells, sometimes called the finely granular cells of Schultze, are 
from two to three times the size of the red cell (10 to 12 microns) in diameter, 
the size depending upon the extent to which the cell is flattened by pressure. 
These cells are always smaller than the large mononuclear type and quite 
small specimens are frequently found in cases of myeloid leukemia. They 
are the most sharply characterized cells of the blood, their protoplasm being 
relatively great and showing slightly acidophilic staining properties. The re- 
ticular portion of the protoplasm is very slightly basophilic, showing occasionally 
nodal thickening or granules when stained with the methylene blue dyes. 
Throughout this protoplasm are scattered fine dust-like granules, the e granules 
of Ehrlich, which are not all of the same size and which stain with the neutral 
principle of the dyes. In some cases these granules may gradually diminish 
so that they may be apparently absent or at least undetected. The triple 
stain is the characteristic stain for these granules, showing them of a distinct 
lilac color; but various other acid dyes, such as eosin, give various tints to them, 
so that they are recognizable as distinctly reddish granules in specimens stained 
by any of the Romanowsky modifications. The nuclei are elongated and 
constricted and may appear in the form of a bent rod or a mass of interwoven 
fine fibers, showing a thin chromatin-rich structure, or a distinctly S-shaped 
formation with oval thickenings. Frequently these nuclear masses appear 
as if separated into two or more distinct nuclei, but as a rule these masses 
are connected by fine bands and should, therefore, be considered rather as 
polymorphous than as polynuclear. The nucleus shows a reticular structure 
with nodal thickenings and is very basophilic. These forms constitute from 
65 to 75 per cent, of the total number of cells, their absolute number averaging 
5,000 per cmm., the percentage ranging from 20 to 40 per cent, in the first 
years of life when the lymphocytes are increased. When outside of the 
blood-vessels they form the usual pus-cells and are extremely active phagocytes. 
These cells are derived from the mononuclear neutrophilic myelocytes which 



THE BLOOD. 505 

are normal habitants of the bone-marrow. They run their course as such 
and are not transformed into other cells, their granules diminishing in patho- 
logical conditions or with age, such change being associated with degeneration 
in the nucleus. 

Perinuclear Granulation. 
Sometime ago Neusser reported a finding of basophilic granules in 
certain leucocytes, especially the mononuclear and polynuclear type. These 
granules surrounded the nucleus and even appeared attached to it, showing 
a variable size and being more or less refractive. He regarded these perinuclear 
granulations as characteristic of the uric acid diathesis, but the work of Futcher 
and of Simon show that these granules are undoubtedly artefacts which can 
be produced by heating and by variation in the staining. 

Arneth's Classification. 

Arneth l in his study of the blood both in health and disease has been led 
to classify the polymorphonuclear neutrophiles into five classes depending 
upon the number of nuclear lobes. His divisions seem to be fairly constant 
in health, but show great variation, especially in infectious conditions. His 
first subdivision, known as Class 1, is divided into (a) M cells, which are 
mononuclear forms identical with Ehrlich's myelocyte, (b) W cells, representing 
forms which show only a slightly indented nucleus, the indentation never extend- 
ing beyond the middle of the nucleus; this cell forms what is called the meta- 
myelocyte, (c) T cells, in which the indentation of the nucleus is deeper than 
in the W cell, but there is no distinct separation into isolated loops, this form 
constituting the true polymorphonuclear type. The first two varieties of Class 

1 are usually seen only under abnormal conditions, although the W cell 
may be found to the extent of 0.2 per cent, in healthy conditions. The cells 
of Class 1 are usually present, according to Arneth, to the extent of 5 per 
cent., Simon giving this percentage as from 4 to 9. The second class of 
Arneth embraces cells with two distinct nuclear fragmentations and shows three 
subdivisions: (a) 2 K cells, neutrophiles whose nucleus consists of two round 
nuclear portions; (b) 2 S cells, neutrophiles whose nucleus consists of two 
distinct S-shaped forms; (c) 1 K 1 S cells, neutrophiles whose nucleus consists 
of one round nuclear portion and one S-shaped division. These cells of Class 

2 constitute, according to Arneth, 35 per cent, of the total neutrophiles, while 
Simon's figures are 21 to 47 per cent. In Arneth's figures we find the 2 S cells 
forming about 23 per cent, of the total of this class. The third class has three 
nuclear divisions and is subdivided into four parts, as follows: (a) 3 K cells, 
(b) 3 S cells, (c) 2 K 1 S cells, (d) 2 S 1 K cells. Arneth gives the percentage 
of Class 3 as 41, the 2 K 1 S and the 2 S 1 K subdivisions, each representing 
approximately 16 per cent, of the total of this class. Simon's figures for this 
class range from 33 to 48 per cent. The fourth class comprises cells with 
four nuclear divisions showing five subgroups, as follows: (a) 4 K cells, 
(b) 4 S cells, (c) 3 K 1 S cells, (d) 3 S 1 K and (e) 2 K 2 S cells. This class, 
from Arneth's figures, shows a percentage of 17, the 4 K, 3 K 1 S, and 2 K 2 S 

1 Jena, 1904. 



506 DIAGNOSTIC METHODS. 

types being largely in excess of the other forms. The fifth class comprises 
cells with five or more nuclear subdivisions and may be arranged into five 
groups, this class representing about 2 per cent, of the total neutrophiles. 
It is probable that these various classes represent the gradual development 
of the polymorphonuclear neutrophile, the older the cell the greater the tendency 
to reach Class 5, while in conditions associated with new formation of cells, 
as in infectious conditions, we find the percentages of the earlier classes being 
increased, that of the later ones diminished. Minor and Ringer 1 have recently 
emphasized the prognostic value of this method in pulmonary tuberculosis. 

(b). Polymorphonuclear Eosinophiles. 
These cells, sometimes called the coarsely granular cells of Schultze, 
are somewhat smaller than the preceding, varying in size from that of a lympho- 
cyte to that of the neutrophile. Their protoplasm is usually somewhat less 
in amount than is that of the neutrophile and may not be distinct. It is filled 
with coarse, round, or slightly oval granules about one micron in diameter, 
which are very refractile and appear in the fresh specimen distinctly black, 
while in the stained smear they take up the acid portion of the dye and are 
called, therefore, acidophilic, oxyphilic, or eosinophilic cells. 2 These granules 
are the a granules of Ehrlich, and are sometimes associated with the /? granules 
of Ehrlich which are about the same size or a little smaller than the a granules 
and take both the acid and the basic stains, although with the ordinary staining 
solutions they appear, like the a forms, stained with eosin. The nuclei of these 
cells are coarsely reticulated, are larger and thicker than those of the neutrophiles, 
are usually bilobed, and more frequently show distinct separation of these 
lobes. These nuclei do not stain very deeply with the nuclear dyes, so that 
they may be rather indistinct. These cells are probably derived from the 
mononuclear eosinophile myelocytes of the bone-marrow and constitute from 
two to four per cent, of the total number of leucocytes, their average number 
being from 100 to 200 cells. 

(c). Polymorphonuclear Basophiles. 
These cells, frequently called mast cells, resemble the neutrophile cells 
in the fresh specimen, but show quite distinct characteristics in the stained 
form. Their protoplasm is much the same as that of the neutrophile and shows 
the same relation toward the nucleus which does not, however, so frequently 
form distinct lobes as does the nucleus of the neutrophile. The size of these 
cells averages about 10 microns, but shows a marked variation, being very small 
in myeloid leukemia. These cells are characterized by their granules, the 7 
granulation of Ehrlich, which are more irregular in size than are the neutrophile 
granules and are not so extensively scattered through the protoplasm. These 
granules show the peculiar property of metachromasia, being colored red with 

1 Am. Jour. Med. Sc, 1911, CXLI, 638. See, also, Ringer, Ibid., 1912, CXLIV, 561; Kramer, 
New York Med. Jour., 1913, XCVII, 1241; Cummings, Calif. State Jour. Med., 1913, XI, 
286; Cooke, Brit. Jour. Tuberc, 1914, VIII, 211. 

2 See Muller (Wien. klin. Wchnschr., 1913, XXVI, 1025 )for a discussion of the chemical 
properties of these granules. Acton and Knowles (Indian Jour. Med. Research, 1914, 1, 523) 
believe the Kurloff bodies of the bone-marrow to be forerunners of the eosinophile granules. 
See, also, Pappenheim, Folia Haemat., 1914, XVIII, 224; Downey, Ibid., 1915, XIX, 148. 



THE BLOOD. 507 

violet dyes and with the blue dyes violet, although with absolutely pure methyl- 
ene blue they take a blue shading. In cases of myeloid leukemia these granules 
are particularly soluble in water and may, therefore, not be seen if aqueous 
stains are used, while in normal blood the granules are usually water-fast. 
These cells stain best with Ehrlich's dahlia stain, or Turk's iodin solution, 
and take a tone which is not strictly basophilic, but resembles more that of 
mucin, on which account they have been called mucinophiles. Whether these 
granules are all of the same significance is questionable. The true mast cell 
granules are known as the 7 granules, while other basophilic granulations have 
been found by Ehrlich and are known as the delta (5) granules. 1 These latter 
granules are found in the large mononuclear cells, especially in the lymphocytes, 
and do not stain with Ehrlich's dahlia stain. Whether these latter bodies are 
true granules or nodal thickenings is at present uncertain. True mast cells 
probably originate in bone-marrow from a granular mononuclear type corre- 
sponding to other types of myelocytes (Pappenheim). These cells constitute 
about 1/2 per cent, of the total leucocytes, averaging between o and 50 per cmm. 
Ehrlich's dahlia stain for these mast cells is made as follows: 
Distilled water, 100 c.c. 

Saturated absolute alcohol solution of dahlia, 50 c.c. 
Glacial acetic acid, 10 c.c. 

The specimens are heated or fixed by alcohol and are then stained in the 
above stain for 5 to 10 minutes, the mast cells appearing of a distinctly violet 
tone. 

Turk's Iodin Method. 
The specimens are fixed with heat and are then first stained in a 1 per 
cent, alcoholic methylene blue solution, warming the slide very carefully until 
it steams. It is then allowed to cool, washed quickly in water, and dried between 
filter-paper. The slide is then covered with a solution of iodin in potassium 
iodid of the strength 1:300. Allow this solution to act not longer than 1/2 
minute, pour off and mount in the following syrup: 

Iodin, 1 gram. 

Potassium iodid, 3 grams. 

Distilled water, 100 grams. 

Gum arabic q. s. to make a syrup. 
The mast-cell granules appear, when treated in this manner, very distinctly 
outlined and colored black. The nuclei are brownish in color, the erythrocytes 
yellowish-green, and the polychromatophilic erythrocytes dark green, the 
neutrophile and eosinophile granules faintly yellow. 

(C). Leucocytes in Pathological Blood. 

(1). Myelocytes. 

Under this heading we must regard any cell of the bone-marrow as a 
myelocyte, but for diagnostic purposes we have reference more to mononuclear 
cells which are distinctly granular. While the granulations of these cells are 

1 Huguenin (Zentralbl. f. allg. Path. u. path. Anat, 1912, XXIII, 725) reports the 
presence of sudanophile granules in the mast cells in an obscure case. 



508 DIAGNOSTIC METHODS. 

usually either neutrophilic or eosinophilic, we may rarely find, especially in 
myelogenous leukemia, cells which show basophile granulations. The size 
of these myelocytes varies from that of the red blood-corpuscle to that of a 
large mononuclear cell. 

(a). Neutrophile Myelocytes. 
This myelocyte may be either large or small, in the first case being known 
as Cornil's myelocyte (amblyochromatic type), a cell which is much larger 
than the polymorphonuclear leucocyte (at least 15 microns in diameter), and 
showing a round, pale, eccentric nucleus which stains feebly but not diffusely. 
This cell shows many distinct neutrophile granulations and has a narrow zone 
of basophilic protoplasm surrounding the nucleus. It is found almost entirely 
in myelogenous leukemia and has been called by Pappenheim the heteroplastic 
promyelocyte. The second type of neutrophile myelocyte is known as the 
Ehrlich myelocyte (trachyochromatic type of Pappenheim) which is a medium- 
sized cell with a pale central nucleus which stains deeply but not diffusely. 
This cell shows extensive neutrophilic granulation of the protoplasm, which is 
faintly oxyphilic, and has a nucleus which is either perfectly round, oval, or 
indented, but is never lobed nor pycnotic. A distinction between the myelocyte 
and the polymorphonuclear leucocyte should be based entirely upon the structure 
of the nucleus, all those cells with round, oval, or kidney-shaped nuclei which 
occupy at least one-half of the cell and show neutrophile granulations but no 
diffuse staining of the nucleus must be called myelocytes. It is to be remem- 
bered, however, that this type develops into the polymorphonuclear neutrophile, 
so that in abnormal blood we may have all gradations between these two types, 

(b). Eosinophile Myelocytes. 
These cells are exactly analogous to the preceding, with the exception that 
the granules of the more mature form show distinct eosinophilic tendencies. 
The younger forms of these granules may show a purplish-violet or even blue 
color, owing, as Simon states, to the fact that the young eosinophilic granule is 
physically cyanophilic and chemically amphophilic, whereas the mature granule 
is physically erythrophilic, but chemically absolutely oxyphilic. The size of 
these cells is more or less variable, so that it is probable that we have the two 
types of eosinophile myelocytes, corresponding to the large and small neutrophile 
myelocytes. These cells occur more frequently in leukemia, in association 
with tumors of the bone-marrow 1 and in the pseudoleukemic anemia of children. 

(c). Basophile Myelocytes. 
These myelocytes may be of variable size, but are characterized by the 
large centrally located nucleus, which is not clearly defined from that of the 
surrounding slightly basophilic protoplasm. The granules are distinctly 
basophilic and in some cases are very numerous, while in others they may be 
widely scattered through the protoplasm. These cells are practically never 
seen except in cases of severe splenomyelogenous leukemia, in which they may 
reach as high as 47 per cent, with an absolute count of 140,000 cells (Taylor). 

1 von Roznowski (Ztschr. f. klin. Med., 1915, LXXXI, 377) believes that the presence of 
myelocytes in large numbers in cases of cachexia points to metastasis in the bone-marrow. 



PLATE XXI. 



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* 

*• 



IODOPHILIA. 



THE BLOOD. 509 

(2). Irritation Forms. 

These cells vary in size from a lymphocyte to a large mononuclear cell, 
resembling more nearly the former. They are mononuclear, nongranular 
cells, thus differing from the myelocyte which is always granular. The nucleus 
is round and eccentrically placed, showing a very slight chromatin network 
and staining with the triple stain of a bluish-green color while with the Roman- 
owsky dyes the color is a pale blue. The protoplasm is stained a deep brown 
with the triple stain and is thus differentiated from other forms of cells. 
With the methylene blue dyes the protoplasm appears more deeply stained 
than does the nucleus. These cells were first described by Turk and would 
seem to have the same significance as do the myelocytes, namely, an indication 
of marked activity of the bone-marrow. Pappenheim regards them as plasma 
cells and largely derived from the lymphocytes. 

(3). Degenerated Forms. 

Occasionally we find in normal blood degenerated leucocytes which stain 
poorly and show no granules. These may be even so much degenerated that 
they show as the so-called basket cells or ''shadows." This condition is 
very frequently seen in severe infectious diseases, while in the acute leucocytoses 
which occur under many influences diminution in the number of neutrophilic 
granules as well as swelling and fragmentation of the bodies of the leucocytes 
is very common. The changes in the staining qualities of the nucleus seem to 
be the most significant of the lesions in the acute type of degeneration of the 
leucocytes. In chronic degeneration of the leucocytes we find hydropic degen- 
eration, which is frequent in the blood of chlorosis and, when the nuclei are 
involved in this degeneration, seems to be limited to certain cases of leukemia. 
Besides such changes we find fatty degeneration as well as glycogenic degenera- 
tion. This fatty degeneration is characterized by the appearance of fat globules 
in the leucocytes which stain with osmic acid and with Sudan-Ill. For this 
latter reason they have been styled sudanophiles, and have been very carefully 
studied by Buttini and Comesatti as well as by Cesaris-Demel. 

Iodophilia. 

In many pathologic conditions, especially in acute infectious diseases 
and in those associated with all types of sepsis, a so-called glycogen reaction or 
iodophilia may be demonstrated in the bodies of the leucocytes, as well as in 
certain extracellular granules. The technic of this method is as follows: 
An unfixed dry blood smear is exposed to the vapor of solid iodin until it is 
stained a brownish color. After the specimen is stained it is mounted in the 
syrup described on page 507 and is examined with an oil-immersion lens. 
The blood of normal individuals, stained by this method, shows the protoplasm 
of the leucocytes of a bright yellow, while the nucleus takes on a much lighter 
tint. In pathologic blood two types of reaction can be noted. The intra- 
cellular one, which is of greater clinical importance, shows a more or less marked 
diffuse brown color of the entire protoplasm of the leucocyte, or the protoplasm 
contains reddish-brown granules which may be more or less distinct. The 
extracellular reaction is evident in the blood plates, while the intracellular 



5IO DIAGNOSTIC METHODS. 

type is more particularly confined to the neutrophiles, although the mononuclear 
leucocytes may occasionally be tinged brown. Much difference of opinion 
exists as to the nature of this brown-staining substance, Ehrlich regarding 
it as glycogen, while Czerny considers it as an antecedent of amyloid, and 
Goldberger and Weiss regard it as peptone. Kaminer regards this reaction 
as a degenerative change and not as an evidence of regeneration. While 
this reaction has little value in differentiating infectious or septic conditions 
one from the other, it is sometimes of importance in making a diagnosis 
between purulent and nonpurulent affections, being present in the former 
and absent in the latter. These granules have been found by Hofbauer in 
pernicious anemia, secondary anemia, and leukemia, but not in chlorosis or 
pseudoleukemia. This reaction is observed in pneumonia, but is seldom seen 
in tuberculosis, typhoid fever and diphtheria. It is, however, not to be regarded 
as dependent upon infection, as it occasionally obtains in non-infectious 
conditions. 

(D). Differential Counting of the Leucocytes. 

By a differential counting of the leucocytes is meant the counting of the 
different varieties of the leucocytes found in the stained smear and the calcula- 
tion of each type in terms of percentage. The technic of this method is 
that previously outlined and consists in making an even smear upon a glass 
slide and staining it with any of the stains previously mentioned, noting that 
the triple stain does not bring out the granulations of the leucocytes with the 
exception of those of the neutrophiles. 1 It is self-evident that the larger 
the number of leucocytes counted the greater will be the possibility of arriv- 
ing at true percentage relations. It is wise, therefore, to count at least 250 
of these cells, and in many cases, to extend this to 500. If the smear is 
even and the leucocytes well distributed throughout, 100 cells will frequently 
suffice. 

For a differential count a satisfactory classification is an absolute essential. 
As none of the systems at present advanced are entirely adequate, we still use 
the classification of Ehrlich, which is as follows: Small mononuclears, large 
mononuclears (including the transitional), polymorphonuclear neutrophiles, 
eosinophiles, basophiles (mast cells), and myelocytes. The characteristics 
of these cells have been previously given, but may be summed up in this connec- 
tion. By a small mononuclear is meant any nongranular mononucleated cell 
smaller than a polymorphonuclear neutrophile. A large mononuclear is any 
nongranular cell with a round or oval nucleus and larger than a polymorpho- 
nuclear neutrophile. A cell of the same description and size but with an 
indented nucleus is a transitional form. The polymorphonuclears are cells 
which are about 10 microns in size and show a diffuse granulation, which may 
be either neutrophile, eosinophile, or basophile. It should be remembered 
that the mast cells appear as nongranular forms when the triple stain is 
used, so that the characteristics of the nucleus in its relation to the protoplasm 

l See Arneth, Deutsch. Med. Wchnschr., 1913, XXXIX, 2560; also, Dunzelt, Miinch. 
Med. Wchnschr., 1913, LX, 2616. 



Percentage. 


Number per cmm. 


20-25 


1200-2000 


3-5 


200-400 


65-75 


5000 


2-4 


100-200 


0-1/2 


0-50 



THE BLOOD. 511 

must be borne in mind. The percentage relations of these cells in norma) blood 
are as follows : * 

Small mononuclears, 

Large mononuclears, 

Polymorphonuclear neutrophiles, 

Polymorphonuclear eosinophils, 

Polymorphonuclear basophiles, 

In the writer's laboratory differential staining is usually carried out with 
the use of the Wright or Giemsa stain as he has found the usual triple stain 
quite unreliable. Although the granular differentiation is not as distinct as 
could be desired, yet one soon becomes accustomed to the staining of various 
cells so that it is not a matter of great difficulty to distinguish the various types. 
In many cases it is not the easiest matter to distinguish myelocytes from the 
small mononuclear types, but careful study will usually clear up any obscurities 
which exist. 

(£). Number of Leucocytes. 

The normal number of leucocytes in a cmm. of blood has been given 
various figures. As a rule, it may be said that anything above 10,000 leucocytes 
per cmm. should be considered pathological, the normal variation running 
from 5,000 to 9,000 cells. In estimating the normal number of white cells, 
both in health and in disease, a large number of factors which influence these 
cells must be taken into consideration. Thus vasomotor phenomena, varia- 
tions in the volume of the plasma, inflammatory processes, state of digestion, 
age, variations in different parts of the circulatory system, and many different 
disease processes usually bring about an increase in this number, while many 
pathological conditions are associated with a reduction. An increase in the 
number of white cells is usually spoken of as a leucocytosis, but it must be 
remembered that such an increase may be purely physiological and should be 
sharply differentiated from a pathological increase which is clinically the more 
important. This increase is usually referable to the increase in the number 
of the polymorphonuclear neutrophiles, while an increase in the other varieties 
of cells is spoken of as a lymphocytosis, myelocytosis, esoinophilia or an eosino- 
philocytosis, or a mixed leucocytosis. 

A diminution of the number of polynuclear neutrophiles is designated as a 

leucopenia, which is the more usual form in which reduction of the cells occurs. 

It is to be remembered that these conditions may be transitory and symptomatic 

pointing to a purely physiological process, while a more permanent and more 

marked increase or decrease in the number should be considered pathological. 

It is rare that we find the increase limited absolutely to one variety of cell, the 

increase in the others being less marked. The absolute number of these cells 

1 See Bunting, Am. Jour. Med. Sc, 1911, CXLII, 698; Galambos, Folia Hsemat.,1912, 
XIII, 153; von Torday, Virchow's Arch. f. path. Anat., 1913, CCXIII, 529; Mehrtens (Arch. 
Int. Med., 1913, XII, 198) calls attention to the frequency of relatively low polynuclear leuco- 
cyte counts with high lymphocyte counts in the routine clinical work. Warfield (Jour. Am. 
Med. Assn., 1915, LXIV, 1296) advises subdivisions of these groups so that we have seven 
classes. 



512 DIAGNOSTIC METHODS. 

is much more to be regarded than their percentage relation, as with an 
increased number of leucocytes the actual number of some of these varieties 
may be increased, although the percentage may be diminished; while with a 
low leucocyte count the percentage may be increased and the absolute number 
diminished. It is wise, therefore, to report not only the percentage relations 
of the leucocytes, but also the actual number of these cells per cmm. This 
will correct mistaken ideas as to an apparent increase or decrease in any particu- 
lar variety of cell. Thus a differential leucocyte count may show a percentage 
of 50 for the neutrophiles and at the same time an actual number of 10,000 
per cmm., giving rise to confusion as to the actual relation of these important 
leucocytes. 

Leucocytosis (Polymorphonuclear Neutrophiliosis). 
As stated above, anything above 10,000 leucocytes per cmm. should be 
regarded as a leucocytosis. This is, however, relative and should not be 
considered pathological without taking into consideration all of the physiological 
and pathological influences. Regarding the theoretical cause of leucocytosis, 
little is definitely known. The influence of infectious processes is such as 
usually to increase the number of leucocytes in the blood, as an attempt on the 
part of the system to overcome by phagocytosis the action of the bacteria of 
the various diseases. Yet we find in some of these infectious processes, notably 
in typhoid fever, a marked reduction in the number of the white cells, although 
the bacillus typhosus is present in large numbers in the blood at the same time. 
We must, therefore, assume some specific influence upon phagocytosis and 
chemotaxis, as a general infection is not necessarily associated with an increase in 
the number of white cells, but is dependent more upon the specific nature of the 
infection. The work upon opsonins and vaccine therapy may open up an 
entirely new field in our study of this subject. The leucocytosis shown in 
noninfectious conditions is still a matter of much dispute and has probably 
more to do with variations in the plasma than with direct increase in the 
number of white cells. 
Classification. 
According to Limbeck, 1 the following classification of the various leuco- 
cytoses is the most comprehensive: 

(1). Physiological leucocytosis. 
(a). Leucocytosis of digestion, 
(b). Leucocytosis of pregnancy, 
(c). Leucocytosis of the new-born. 
(2). Pathological leucocytosis. 
(a). Inflammatory leucocytosis. 
(b). Leucocytosis associated with malignant tumors (cachectic 

leucocytosis). 
(c). Posthemorrhagic leucocytosis. 
(d). Agonal or antemortem leucocytosis. 
(3). Leucocytosis following medicinal and therapeutic measures. 
(4). Leucocytosis from other causes. 
1 Jena, 1896. 



PLATE XXII 




POLYNTJCLEAR LeUCOCYTOSIS. (WRIGHT'S STAIN.) 



THE BLOOD. 513 

(1). Physiological Leucocytosis. 
(a). Leucocytosis of Digestion. 

Normally a leucocytosis of the polynuclear type will be observed beginning 
about one hour after a meal rich in proteins and will reach its maximum in 
from three to five hours. 1 The actual figure reached varies in different persons, 
being usually an increase of about one-third, the maximum often reaching 
15,000 cells, but more usually not much over 10,000. In this increase the 
small mononuclear cells may be absolutely as well as relatively increased. 
In some persons this leucocytosis of digestion does not appear, which fact may 
be referable to marked torpidity of the intestines, to a prolongation of the 
process of digestion, or to a large absorption of fluids. It has been found that 
a highly albuminous diet has a much more marked influence upon this leuco- 
cytosis than does a diet of vegetables and fat. The rapidity of absorption and 
of digestion must be taken into consideration as factors which influence the 
appearance or nonappearance of the leucocytosis. In children the increase 
in cells is much more marked than in the adult, due probably to the increased 
digestive and absorptive powers, providing the food taken is, as it should be, 
easily digestible and absorbable. 

Various pathologic conditions influence this digestion leucocytosis. Thus 
Miiller has found that in cases of carcinoma of the stomach a digestion leuco- 
cytosis is rarely observed after heavy meals. There are, however, a few cases 
of gastric cancer in which a slight increase has been observed. This failure 
of leucocytosis is probably due, as Schneyer shows, to lessened absorption as 
a result of involvement of the lymphatics rather than to a malignant stenosis 
of the pylorus, as the benign stenoses are usually associated with a leucocytosis. 
That the lack of leucocytosis is due more to diminished absorption than to 
lack of digestive power is shown by the usual occurrence of a digestion leucocy- 
tosis in ulcer of the stomach, chronic gastritis, and in dyspeptic conditions. 
Just why carcinoma, with or without stenosis, should be associated with a 
normal or subnormal number of leucocytes after digestion is hard to explain, 
in view of the fact that these other conditions are associated with a leucocytosis. 
Practically all absorption of food material occurs from the bowel and we 
would naturally expect to find disorders of the intestinal canal leading more 
frequently to a normal leucocyte count than to a leucocytosis. Very little 
detailed work has been done upon the influence of enteric troubles upon the 
leucocyte count, but the writer has seen several cases in which a digestion 
leucocytosis did not occur and in which the findings both ante- and postmortem 
were entirely related to the bowels. The examination of the blood may, how- 
ever, clear up a diagnosis of carcinoma, but will not always permit of a differen- 
tial diagnosis between this condition and pernicious anemia. It is to be 
remembered that a leucocyte count must be frequently made in order that 
this test may be of any value whatever and that a considerable rise only is to 
be taken as evidence of a leucocytosis. Patients with cancer either of the 
stomach or of other viscera frequently show a leucocytosis as the result of 

1 See Brasch, Ztschr. f. exp. Path. u. Therap., 1912, X, 381; also, Mitchell, Am. Jour. Dis. 
Child., 1915, IX, 358. 

33 



514 DIAGNOSTIC METHODS. 

cachexia and this should be remembered in the interpretation of a leucocyte 
count following digestion. 

(b). Leucocytosis of Pregnancy. 

It has been shown that from 50 to 75 per cent, of cases of pregnancy are 
associated with a leucocytosis, averaging about 15,000 cells per cmm. This 
is especially true of primiparae, but is often shown in the multiparas Just 
what cause can be given for this rise is uncertain. It is more than likely 
that a condition of slight intoxication is present due to the overloading of 
the blood of the patient with the products of metabolism of the fetus. These 
substances are not normal to the blood of the woman and in consequence act 
as foreign bodies which may attract, by their chemotactic influence, the white 
cells and bring about an increase. As these cases of pregnancy show an ab- 
sence of a digestion leucocytosis it has been assumed that the increase in 
leucocytes is due to a prolonged digestion leucocytosis, but this does not seem 
probable. The changes in the breasts and in the uterus during this period 
have suggested these organs as the factors influencing this leucocytosis, but 
such inferences do not seem to be directly warrantable. The leucocytosis 
of pregnancy is a mixed leucocytosis, all of the various types of leucocytes, 
with the exception of the eosinophiles being increased. After the birth of the 
child the leucocytes gradually diminish in number and normally reach their 
usual values in from four to fourteen days after delivery. Should complica- 
tions, such as postpartum hemorrhage or septic fever arise, the leucocyte 
count may remain high until these complications have subsided. In multi- 
paras these changes in the number of leucocytes are not so marked as in the 
case of the primiparae but a slight rise always occurs. This fact has been 
attributed to the lessened reactivity of the organisms to the influence of the 
toxic substances thrown into the blood from the cells of the fetus. 

(c). Leucocytosis of the New-born. 

As Askanazy has found, the blood of the fetus shows a diminution in the 
number of leucocytes, owing to the fact that there is no function as yet estab- 
lished for these cells in utero. In the blood of the new-born, however, a leuco- 
cytosis running from 15,000 to 20,000 cells may be observed, these figures 
going as high as 40,000 under the influence of the first feeding. As the weight 
of the child begins to diminish these cells are markedly reduced in number 
to about 8,000, and are subsequently increased to 10,000 as the child begins 
to gain in weight. The high leucocyte count at birth is more probably due 
to the rapid blood formation than to a concentration of the blood, although 
this latter factor as well as that of the influence of digestion must be taken 
into consideration. The increase in the number of cells is chiefly limited 
to that of the small mononuclear cell, the differential count of the leucocytes 
showing during the early periods of life from 40 to 60 per cent, of the total 
number. This leucocyte count, if taken at the moment of birth, will not, 
however, vary much from that of the adult, the change becoming more marked 
in the early periods of life. 



THE BLOOD. 515 

(2). Pathological Leucocytosis. 
(a). Inflammatory Leucocytosis. 

In most cases of inflammatory nature, of acute infections and general 
febrile diseases, there is observed an absolute increase of the polymorphonuclear 
neutrophiles,, which runs more or less parallel to the temperature. This 
increase may run from 10,000 to 50,000 cells and diminishes as the influence 
of the inflammatory process is diminished. In general it may be said that 
leucocytosis represents the reaction of the individual to the disease. A high 
count may mean a vigorous reaction to the infection; a low count may mean 
either a poor reaction and hence an unfavorable condition of the patient, or 
it may indicate a very mild degree of infection with a normal reactivity of the 
patient. It must be said that all diseases of an infectious nature are not 
necessarily associated with a leucocytosis. For instance, pneumonia shows 
a leucocytosis which runs parallel to the degree of virulence; measles, influenza, 
malaria, and tuberculosis are rarely if ever associated with a leucocytosis 
unless complications arise or the conditions become very severe. In typhoid 
fever we usually find a leucopenia which, however, is associated with a relative 
lymphocytosis; if complications such as perforation arise a leucocytosis may 
appear. Among the conditions causing leucocytosis are acute lobar pneu- 
monia, the count running between 20,000 and 100,000, depending upon the 
severity of the infection and the degree of resistance against the infection. 
Acute articular rheumatism, diphtheria, acute cerebrospinal meningitis, 
follicular and suppurative tonsillitis, scarlet fever, mumps, rabies, erysipelas, 
ulcerative endocarditis, small-pox, cholera, general pus infections of the serous 
membranes and of the mucous membranes, 1 acute bronchitis, and many other 
conditions cause a leucocytosis of the polymorphonuclear type. The variations 
in these many conditions will be discussed under the Pathology of the Blood. 

The exact cause of this leucocytosis following inflammatory processes 
is still a matter of much discussion. The bone-marrow has been shown to be 
markedly increased as regards cellular proliferation in the early stages of the 
inflammation and may be so markedly changed as to cause permanent de- 
rangement in the functions of this blood-forming organ. That leucocytosis 
has much to do with immunity both from the standpoint of phagocytosis 
and from that of Ehrlich's side-chain theory cannot be questioned. 2 A leuco- 
cytosis must represent the attempt on the part of nature to rid the blood and 
the system of the bacterial and toxic products of the disease. Whether phago- 
cytosis as such or under the influence of various opsonins is the direct cause 
of immunity and of recovery from any specific infection must be left to a later 
chapter. 

(b). Cachectic Leucocytosis. 

This variety of leucocytosis is the least uncertain. The cases of carcinoma 
and of sarcoma show a leucocytosis which is not definite for the particular 
kind of cancer, but is more usual with the latter than with the former type. 

1 See Smith, Surg. Gynec. and Obst., 1913, XVI, 403. 

2 Gay and Claypole (Arch. Int. Med., 1914, XIV, 662) call attention to the specific hyper- 
leucocytosis following inoculation with typhoid vaccines. 



516 DIAGNOSTIC METHODS. 

The leucocytosis is usually one of the polymorphonuclear type, but is frequently 
associated with an increase in the number of mononuclear cells. This leuco- 
cytosis has no direct relation with the situation of the tumor and is not always 
present in all cases of cancer. Whether this leucocytosis be due to an intercur- 
rent infection is a question which must be left for more detailed work, but it 
would seem wise to accept Ewing's statement that a marked leucocytosis in the 
course of a cachexia, from tertiary syphilis, tuberculosis, nephritis, and in the 
majority of cases of carcinoma, should suggest a search for a complicating 
infection, while in the sarcomata a leucocytosis is much more common as a 
direct result of the disease. 

(c). Posthemorrhagic Leucocytosis. 

A well-marked leucocytosis, which may begin in from ten to fifteen min- 
utes and may reach as high as 20,000 cells within an hour, has been often ob- 
served following extensive acute hemorrhages. The leucocytosis in these cases 
bears a general relation to the extent and rapidity of the loss of blood and 
usually disappears or diminishes long before regeneration of the blood has 
occurred. 1 This leucocytosis is of the polymorphonuclear type and is referable 
rather to the sudden outflow of lymph which occurs as a compensation for the 
loss of fluid than to a new production of cells, as this latter process does not 
take place for some time. In hemorrhages which are slight and long-continued 
as in cases of gastric or intestinal ulcer, the duration of the leucocytosis as well 
as its extent is very brief. Stassano and Billou assume from their work that 
a hypoleucocytosis follows a severe hemorrhage while a true leucocytosis is 
observed after the loss of small quantities of blood. These findings seem to 
be rather doubtful, except where the hemorrhage has been so extensive as to 
cause death. 

(d). Antemortem Leucocytosis. 

This form of leucocytosis has been questioned, especially by Arneth, 
but there seems to be little doubt that such a leucocytosis may occur if death 
does not take place too rapidly. In some diseases the leucocytes do not fall 
in number, but in some a distinct rise is noted which has been attributed by 
Ehrlich to the accumulation of white cells along the periphery of the blood- 
vessels as a result of slowing or stasis of the circulation. This type of leuco- 
cytosis is usually of the polymorphonuclear variety and may support the view 
of Limbeck that antemortem leucocytosis, when it does occur, is the result 
of a terminal infection, although it cannot explain those cases which show a 
lymphocytosis rather than the ordinary leucocytosis. The character of the 
antemortem leucocytosis must depend largely upon the precedent condition 
and will be associated with antemortem dissemination of bacteria, ante- 
mortem hyperpyrexia, vasomotor paralysis, serous exudation, etc. 
(3). Leucocytosis Following Therapeutic Measures. 
(a). Drugs. 

It has been found following administration of tonic drugs, ethereal oils, 
myrrh, turpentine, camphor, peppermint, quinin, and other drugs, that a 

1 See Levison, Jour. Am. Med. Assn., 19 15, LXIY, 1294. 



THE BLOOD. 517 

leucocytosis of a more or less extent occurs, which is probably referable to 
the same cause as is digestion leucocytosis. Many of these drugs if applied 
locally for the purpose of counterirritation were shown to have the same 
effect. Extracts of tissues, especially those containing large amounts of 
nucleinic acid and of purin substances, produce an extensive leucocytosis, 
which fact has been taken advantage of in the administration of the nuclein 
substances as therapeutic remedies. In the case of those drugs which destroy 
the red cells, such as the coal-tar antipyretics, chlorates, and illuminating 
gas, a normal number of white cells is usually observed, although Simon wrongly 
states that a hyperleucocytosis follows the use of such drugs. After the pro- 
longed use of chloroform or ether a polynuclear leucocytosis is generally 
observed which is usually of short duration. In this connection it is well to 
remember that an increase in the number of leucocytes of 10,000 to 15,000 above 
the normal value of the individual should be regarded as evidence of infection 
if this increase is sustained for more than a few hours. 

(b). Baths. 
After a cold bath the leucocytes of the polynuclear variety have been 
shown to be increased from 100 to 300 per cent. 1 This is true only if the bath 
is of moderate duration, a prolonged cold bath taken to the point of exhaustion 
will diminish rather than raise the number of white cells. Experiments on 
the result of hot baths have shown just the reverse condition, namely, a hot 
bath of short duration produces a decrease, while one of long duration causes 
an increase in the number of white cells. Massage in itself following either 
a hot or a cold bath tends to increase the number of leucocytes. 

(c). Exercise. 
Prolonged muscular exercise, as taken in the form of gymnasium work, 
as applied in the various therapeutic movement treatments, and also as given 
in the more violent athletic contests produces a rise in the number of leucocytes 
which is temporary and is characterized more by an increase in the number of 
polynuclear cells than of any other variety of the leucocytes. 

(4). Leucocytosis from Other Causes. 

Cyanosis, passive hyperemia obtained by the method of Bier, shock 
whether physical or mental, injection of various toxins, such as Koch's tuber- 
culin, the various autovaccines, and injection of various organic principles, 
such as peptone, pus, organ extracts, etc., have all been shown to produce a 
polynuclear hyperleucocytosis, either by causing stasis of the circulation or by 
increasing the chemotactic power of the serum. 2 The exact explanation of 
chemotaxis is still in doubt and we find that some substances are positively 
and others negatively chemotactic, thus accounting for the lack of phagocy- 
tosis in certain conditions, such as pneumonia, although in these same condi- 
tions we may have a leucocytosis as a direct result of an infection. From the 
work of Rosenow we learn that the phagocytic action of the leucocytes in 

1 See Rovighi and Secchi (Rif. Med., 1914, XXX, 617; Munch. Med. Wchnschr., 1914, 
LXI, 1 721) for a discussion of leucocytosis following exposure to cold. 

2 SeeVeraguth and Seyderheim (Munch, med. Wchnschr., 1914, LX[, 301) for the leucocy- 
tosis following use of electricity. 



518 DIAGNOSTIC METHODS. 

pneumonia is increased by the addition of an extract of the active pneumo- 
cocci; whether this extract increases the chemotactic power of the bacteria in the 
blood or whether certain restraining influences are overcome is still unsettled. 
Future work upon phagocytosis and upon the factors governing increased op- 
sonic power in the various infections may show us what causes are effective in 
such conditions. 

In the previous discussion of leucocytosis we have had reference more 
to an increase in the number of polynuclear cells, this type being more properly 
called polynuclear hyperleucocytosis. It is very rare that this condition 
exists in the absolutely pure state. 

Mixed Leucocytosis. 

By this is meant an increase of both granular and of nongranular cells 
of various types, its more common interpretation, however, being a leucocytosis 
characterized by an increase in the number of neutrophile myelocytes. This 
neutrophilic myelocytosis is best seen in leukemia in which the absolute number 
of myelocytes may reach 150,000, the increase in no other condition, according 
to Ehrlich, rising above 1,000 cells. The increase in the myelocytes in leuke- 
mia is not limited to those of the neutrophile variety, but is commonly associated 
with a marked increase in the number of eosinophile types as well as with an 
increase in the number of mast cells. In pernicious anemia we may also rind 
a large increase in the number of these myelocytes. These cells when in- 
creased in number are clinically significant of exhaustion of the bone-marrow, 
but should never be interpreted in this way unless they remain while the leu- 
cocyte count is falling; in other words, they are significant only when constitut- 
ing a large percentage of the leucocytes. 

Lymphocytosis. 

A relative or absolute increase in the number of lymphocytes is of fre- 
quent occurrence and has occasionally some significance. In judging of this 
condition the actual number of cells present as well as their numerical rela- 
tions to the other varieties of leucocytes must be considered. The term 
lymphocytosis must always be applied to an absolute increase in the number 
of these cells, the normal number being between 1,200 and 1,500 per cmm. 
Physiologically we find this condition in infants and during a digestive leucocy- 
tosis in the adult. 1 The child shows at birth from 50 to 65 per cent, of these 
cells which percentage gradually diminishes, reaching the normal percentage 
(20-25) about the age of puberty. 

Pathologically this condition is observed in poorly nourished children, 2 
those showing the "constitutio lymphatica," rachitis, whooping-cough, gastro- 
intestinal disturbances in the child, cervical adenitis, splenic tumors, in most 
infectious diseases of children, 3 and especially in lymphatic leukemia, in which as 
high as 90 per cent, of a largely increased leucocyte count is referable to the 
small lymphocytes. In the splenomyelogenous form of leukemia and following 

1 Kaufmann, Jour. Am. Med. Assn., 1914, LXIII, 1104; Staines, James and Rosenberg 
(Arch. Int. Med., 1914, XIV, 376) report a lymphocytosis due to increased altitude. 

2 See von Hoesslln, Munch, med. Wchnschr., 1913, LX, 1129 and 1206. 

3 See Marchand, Deutsch. Arch. f. klin. Med., 1913, CX, 359. 



THE BLOOD. 519 

removal of the spleen we observe a steady increase in the number of lymphocytes 
continuing during the first year. It must be remembered that a leucocyte 
count may be low and yet a lymphocytosis exist. This is shown in typhoid 
fever, amebic dysentery, chlorosis, pernicious anemia, scurvy, and other 
conditions in which the leucocyte count is low, the granular cells being dimin- 
ished in number, but the lymphocytes being increased. 1 In the congenital and 
also secondary acquired syphilis we find a lymphocytosis which must be re- 
ferred to involvement of the lymph-glands. The worker must not be led into 
a mistaken interpretation of his blood-findings, as enlarged lymph-glands 
may give the same picture of lymphocytosis as is seen in Hodgkin's disease, 
chronic and acute lymphatic leukemia. 

Eosinophilia. 

By this is meant an absolute increase in the number of eosinophilic cells. 
The average number of these cells is between one and two hundred per cmm., 
hence the term eosinophilia should be limited to those cases showing counts 
something above 250. We find a physiological eosinophilia during childhood, 
the average increase being about 1 to 2 per cent, above the normal adult find- 
ing. No physiological relations have been established between eosinophilia 
and sex, pregnancy, menstruation, digestion, or old age; however racial distinc- 
tions are sometimes shown by eosinophilia, the natives of southern China 
showing between 15 and 20 per cent, of the leucocytes as eosinophiles. 

Pathological Eosinophilia. 

We observe variations in the number of eosinophiles in various affections 
of the bone-marrow. Thus in splenomyelogenous leukemia, we may find 
these cells increased as high as 30,000 per cmm. According to Ehrlich, a 
diagnosis of this form of leukemia is warranted only when we have an increase 
in the number of eosinophiles, but it cannot be doubted that cases are relatively 
frequent in which these cells are not increased. In sarcoma of the bone-mar- 
row as well as in osteomyelitis and osteomalacia, we find these cells usually 
increased in number. In chlorotic conditions we may find the eosinophiles 
moderately increased, while in secondary anemias especially those following 
infection with parasites a marked eosinophilia may be observed. After ex- 
tirpation of the spleen as well as in cases of chronic splenic tumor we find an 
eosinophilia which may run from 10 to 40 per cent, higher than the normal, 
constituting as high as 40 per cent, of the total number of leucocytes. Whether 
or not a pure disease of the lymph-glands is associated with the eosinophilia 
is unsettled, but if metastases have extended to the bone, an eosinophilia of 
marked degree is usual. 

True bronchial asthma is always associated with an increase in the num- 
ber of eosinophiles, one case of Billings showing 54 per cent, of the total leuco- 
cytes as eosinophiles. This relationship in asthma is of importance from a 

1 Borchardt (Deutsch. Arch. f. klin. Med., 1912, CVT, 182) shows that a lymphocytosis 
obtains in a large majority of cases of disease of the thyroid, hypophysis and suprarenal gland. 
This is oftentimes associated with a leucopenia. Cabot (Am. Jour. Med. Sc, 1913, CXLV, 
335) calls attention to the occasional occurrence of a lymphocytosis in infections usually asso- 
ciated with a polynucleosis. See, also, Huhle, Deutsch. Arch. f. klin. Med., 1914, CXIII, 455. 



520 DIAGNOSTIC METHODS. 

diagnostic standpoint as asthmatic attacks from other causes are not asso- 
ciated with an eosinophilia. It is true that in emphysema a marked eosino- 
philia does occur so that we may find, in cases in which this condition complicates 
a tuberculosis, that an eosinophilia obtains. Tuberculosis . of the lungs or 
of other tissues does not show an eosinophilia unless complicated by emphy- 
sema, cachexia, or secondary infection. 1 

A large number of skin diseases are associated with eosinophilia, the highest 
count being reported by Zappert in a case of pemphigus showing 4,800 cells per 
cmm. The occurrence of this condition in skin diseases depends not so much upon 
the nature of the lesion as upon its extent, intensity and lack of healing tendency. 
Many skin lesions are known to be produced by toxic agents which have special 
chemotactic influences over the eosinophile cells; this fact may account for the 
eosinophilia in such conditions. The most usual skin diseases showing this 
increase are pemphigus, eczema, psoriasis, urticaria, purpura, scleroderma, 
lupus, leprosy, herpes zoster, and general gouty affections. 2 

It is a general rule that the infectious fevers are not associated with eosino- 
philia, but with the acute polynuclear leucocytosis. 3 In scarlet fever, however, 
we find that eosinophiles are frequently markedly increased, while in acute 
rheumatism and in malaria these cells are usually present in more or less 
increased numbers. 4 In gonorrheal infections the eosinophile leucocytes are 
very frequently marked in the discharge in the early days of infection, diminish- 
ing in number as the number of neutrophiles increases, and increasing again as 
the discharge clears up. It is generally believed that the eosinophile cells 
in the blood are increased coincidently with their increase in the gonorrheal 
discharge. It has been shown that the eosinophiles are usually increased 
in all forms of ovarian disease with the exception of cancer. In syphilis a 
uniform increase of the eosinophiles has been observed, but some cases do not 
show such a regular finding. 

Outside of the splenomyelogenous form of leukemia and the true bron- 
chial asthma, infection with various parasites is accompanied by the most 
pronounced eosinophilia. Any parasite from the harmless pin-worm to 
the most malignant uncinaria may cause an eosinophilia (Emerson). This 
eosinophilia is not necessarily constant nor does its extent bear any relation 
to the severity of the infection. A differential diagnosis between typhoid fever 

1 See Arneth (Deutsch. Arch. f. klin. Med., 1912, CVIII, 323), for the relations of the 
eosinophiles in pneumonia. Chuprin (Med. Obozr., 1913, LXXLX, 17) believes that eosino- 
philes gradually increase in the blood in favorable cases of tuberculosis; also, Brosamlen, 
Deutsch. Arch. f. klin. Med., 1914, CXV, 146; Peppard, Journal-Lancet, 1915, XXXV, 478. 

2 Lifschiitz, Monatsschr. f. Kinderhkde., 1914, XII, 603; Aschenheim, Ztschr. f. Kinder- 
hkde., 1914, X, 503; Putzig, Ibid., 505; Engman and Davis, Jour. Cutan. Dis., 1915, XXXIII, 

73- ' 

3 Schlecht and Schwenker (Deutsch. Arch, f . klin. Med., 1912, CVIII, 405) show the rela- 
tionship of eosinophilia to anaphylaxis. See Herrick, Arch. Int. Med., 1913, XI, 165; also, 
AM and Schittenhelm, Ztschr. f. d. ges. exper. Med., 1913, I, in; Steiger and Strobel, Zen- 
tralbl. f. inn. Med., 1913, XXXIV, 1073. Leopold (New York Med. Jour., 1914, C, 225) 
finds an eosinophilia in chorea. Besancon and Moreau, Ann. de Med., 1914, II, 85; Johns, 
Newi Orleans Med. and Surg. Jour., 1914, LXVII, 453; Weinberg and Seguin, Ann. de l'lnst. 
Pasteur, 1914, XXIII, 470; Herrick, Arch. Int. Med., 1914, XIII, 794. 

- 4 See Ebbell (Norsk Mag. f. Laegevidensk., 1912, LXXIII, 617) for a very naive theory of 
the relation of eosinophilia to the parasitic diseases. 



THE BLOOD. 521 

and trichinosis is frequently possible on the basis of the marked eosinophilia 
in this latter condition, 1 although eosinophilia does not always obtain here. 
Mast-cell Leucocytosis. 

In myelogenous leukemia we find these cells increased to a very large 
extent, often outnumbering the eosinophile cells. An increase of these cells 
is generally regarded as the sole isolated pathognomonic sign of this disease. 
Their increase may be as high as 20 per cent, of the leucocytes. These cells 
have been found in a few cases of cancer, tuberculosis, syphilis, and other 
lesions of the skin, while in bone disease complicated by septic infection a few 
reports of positive findings have been made. 
Leucopenia. 

This is a condition characterized by a reduction either in the total number 
of leucocytes or in one or more groups of leucocytes. The most usual condition 
showing a leucopenia is typhoid fever in which we find the polynuclear cells 
markedly diminished, thus lowering the leucocyte count, while the large 
mononuclear cells are relatively increased. Anything below 5,000 cells is 
regarded as a leucopenia. It must be considered in this connection that the 
typhoid leucopenia remains only when the disease is limited to the intestinal 
canal ; when other organs become involved a leucocytosis supervenes. The count 
in typhoid fever may run as low as 2,000 cells, while in tuberculosis of the 
lymph-glands it may reach 500 cells. In cases of starvation or malnutrition 
the leucocyte count is always low and in chronic intoxication with the heavy 
metals, morphin, alcohol, and cocain, we observe a very low count, as a rule. 
A general rule to be observed in typhoid fever is that an increase in the number 
of white cells following a low leucocyte count is evidence of complications or 
of a mistaken diagnosis. It must be remembered that a relapse in typhoid 
may bring on a hyperleucocytosis, but this must occur during the afebrile 
period, otherwise a leucopenia will remain. 2 In measles, also, we observe a 
leucopenia following the eruption and a leucocytosis preceding the eruption. 
This leucopenia affects the polynuclear neutrophiles while the lymphocytes 
are relatively increased, with disappearance of the eosinophiles. In cases 
of uncomplicated influenza we usually find a diminution in the number of, 
leucocytes, although a normal number may obtain. This enables one to dif- 
ferentiate influenza from pneumonia, in which latter condition a marked 
leucocytosis is practically always present. 3 In pernicious anemia and in 
splenic anemia we find a marked leucopenia during the active periods of the 
disease, the count running, as a rule, between two and three thousand cells. 
(F). Variations in Infancy and Childhood. 

In the first few days after birth the leucocytes may reach as high as 20,000, 
while in the nursing period the cells average 13,000. In this connection the 
influence of a digestion leucocytosis must be remembered as the cells may 

1 Cooper (Wisconsin Med. Jour., 1914, XII, 365) reports a case of trichinosis with 72 per 
cent, of eosinophiles. 

2 See Schneider (Deutsch. Med. Wchnschr., 1915, XLI, 393) for a discussion of leucopenia 
following typhoid inoculation. 

3 Camp and Baumgartner (Jour. Exper. Med., 1915, XXII, 174) show that severe 
inflammatory processes may be associated with a leucopenia. 



522 DIAGNOSTIC METHODS. 

reach as high as 25,000 of which the lymphocytes constitute about 60 per cent. 
As the age of the child increases, the number of leucocytes gradually diminishes 
until the age of puberty, when the average number is about 8,000. As the 
development of the child progresses, the number of lymphocytes is gradually 
diminished and that of the neutrophiles correspondingly increased. The 
relations between the lymphocytes and the neutrophiles must be constantly 
remembered in making a blood count, as the percentage of lymphocytes 
is high at first and then gradually diminishes until the age of puberty; the 
neutrophiles during the same period are at first low, reaching the normal 
65 to 70 per cent, about the age of 15. There are no marked differences in 
the morphology of the granular and of the nongranular cells of the child as 
compared with those of the adult. The characteristic changes in the leucocytes 
of the blood of the growing child are more related to the changes consequent 
upon development and show no signs of degenerative changes. 

(G). Functions of the Leucocytes. 

The functions of the leucocytes are largely related to their powers of 
overcoming the effects of bacterial processes. These results are accomplished 
both through their ameboid powers and their characteristic property of phago- 
cytosis. This latter property is largely influenced by chemotaxis as well as 
by the indefinite increase of so-called opsonins. Just what we are to regard 
as the basis of the opsonic index of the leucocyte is unsettled, but it is certain 
that the same leucocyte may show marked variations in its power of absorbing 
bacteria of different types. Whether this opsonic power is related in any 
way to the various assumptions embraced by Ehrlich's side-chain theory must 
be left for a later section. Besides the above important functions of the 
leucocytes, these cells show oxidizing, reducing, and fermentative powers, all 
of which are of more or less importance in the study of immunity and of reaction 
to bacterial infection. As stated (p. 501), the oxygen catalysts of the blood 
include oxidase, peroxidase, catalase and hemoglobin. To these ferments 
we owe many chemical tests for blood and pus, which are destroyed if the speci- 
men be previously boiled, while hemoglobin is still capable of inducing certain 
oxidations. Kastle and Amoss 1 have studied the peroxidase activity of the 
blood in health and disease, while Kastle 2 has made an exhaustive study of the 
oxidases. Winternitz and Meloy, 3 Winternitz and Pratt, 4 and, especially, 
Winternitz, Henry and McPhedran 5 have turned their attention to catalase. 
The technic of these latter workers is as follows: 0.025 c.c. of blood is taken 
from a puncture of the ear by means of a specially graduated pipet. This is 
immediately diluted with 10 c.c. of distilled water, giving a dilution of 1 1400. 
Five c.c. of this diluted blood are placed in each of two 100 c.c. salt-mouth bottles, 
one being used for the test, the other being held as a check. In one is placed a 

1 Bull. 31, Hyg. Lab. U. S. Pub. Health and Mar. Hosp. Serv., 1906. 

2 Bull. 59, Hyg. Lab. U. S. Pub. Health and Mar. Hosp. Serv., 1909. See Schultze, Munch, 
med. Wchnschr., 1909, LVT, 167; Dunn, Jour. Path, and Bacterid., 1910, XV, 20; and Klopfer, 
Ztschr. f. exper. Path. u. Therap., 1912, XI, 467. 

3 Jour. Exper. Med., 1908, X, 759. 

* Jour. Exper. Med., 1910, XII, 1 and 115. 

6 Arch. Int. Med., 19 11, VII, 624. See, also, Rohdenburg, New York Med. Jour., 19 13, 
XCVII, 824; Waentig and Gierisch, Fermentforsch., 1914, I, 165. 



THE BLOOD. 523 

small vial containing 5 c.c. of neutralized commercial hydrogen peroxid (3 
per cent.). The large bottle is connected with a gas buret, to collect the gas 
formed by the action of the catalase upon the H 2 2 . The bottle is agitated 
for one minute, readings being taken every 15 seconds. Eighty per cent, of the 
normal cases studied show a liberation of 14 to 17 c.c. of oxygen in 15 seconds, 
this being constant for the same individual for a period of many months. 
The importance of this test rests in the variations from day to day or in any 
change from the normal activity of the individual's blood. The amount of 
gas liberated is, therefore, a personal rather than a general factor. Tscher- 
noruzki 1 has recently shown that 1 gram of leucocytes is capable of liberating 
312.9 c.c. of molecular oxygen under the influence of catalase. 

The diagnostic value of this test in disease is as follows: In typhoid 
fever there is no change in the early stages, but toward the third week there is a 
gradual fall accompanying the anemia. During the course of lobar pneumonia 
a slight decline may be noted. Diabetes mellitus shows no change. In 
diseases of the thyroid, the catalytic activity is not constant from day to day, 
in hyperthyreosis a tendency to increase being noted while in hypothyreosis a 
lower level than normal is observed. In renal disease we find no change as long 
as no marked renal insufficiency obtains, but the activity becomes irregular 
from day to day and, as uremia approaches, becomes much lower than normal. 
With retention of urine due to obstruction of the lower urinary tract a marked 
decline is observed. Little change is noted in cardiac cases even in the severe 
types. The toxemias of pregnancy are separable into two classes by this test; 
without renal involvement eclampsia shows no change but, when renal in- 
sufficiency obtains, a marked decline prevails. This test should be of much 
value in diagnosis. 

(7). Blood-plates. 
(a). Appearance. 

These bodies which have been called "third corpuscles" are probably 
not true cellular entities. Hayem has considered them as the direct forerun- 
ners of the erythrocytes and has styled them, therefore, "hematoblasts." 
These bodies have been variously called plaques by Osier, blood-plates by 
Bizzozero, and by Arnold fragments of cells. 2 These so-called corpuscles 
are small colorless bodies containing no hemoglobin. They are about 3 
microns in diameter, round, oval, or rod-shaped without any biconcavity. 
They appear bluish, homogeneous or occasionally granular, and stained 
lightly by both basic and acid dyes. They do not contain any nucleus or 
membrane and become hyaline and glassy as soon as removed from the 
vessel, but on standing they become pale and unite to form granular masses. 
They are very sticky and adhere very extensively to one another, forming 
masses from which fibrinous threads radiate. This fact has lead to the belief 
that they have an important part in the formation of fibrin. (See Duke. 3 ) 

1 Ztschr. f. physiol. Chem., 19 11, LXXV, 216. 

2 See Downey, Folia Haemat., 1913, XV, 25; Brown, Jour. Exper. Med., 1913, XVIII, 278; 
Winogradow, Folia Hsmat., 1914, XVIII, 207. 

3 Jour. Am. Med. Assn., 1910, LV, 1185; also, Arch. Int. Med., 1912, X, 445. 



524 DIAGNOSTIC METHODS. 

Specimens of the platelets are best obtained by puncturing the tip of 
the finger or the ear through a 10 per cent, solution of sodium metaphosphate. 
In this way the blood becomes at once mixed with the fixing fluid and the drop 
may then be placed on a slide and covered with a cover-glass. It has been 
customary to call anything a platelet which is smaller than a red-blood cell 
and which does not contain hemoglobin. The term platelet, however, should 
be reserved more particularly for those bodies which have a peculiar bluish 
refractility, no nucleus, show marked cohesive properties, and soon disintegrate. 

(b). Size. 
The normal size of the blood-platelet averages about 3 microns, although 
Preisich and Hein have reported them as high as 7 microns. As a rule, their 
size varies inversely as their number. Some of these bodies show clear areas 
either in the center or on one side, or on the whole periphery; others become 
crescents, triangles, spindles and threads (Emerson). 

(c). Number. 
The normal number of platelets per cmm. is approximately 250,000, 
this number varying in the same person at different times of day. The physio- 
logical factors influencing the number of these cells are not well understood 
and in pathological conditions we may find large or small numbers of these 
cells. It is generally accepted that they are increased in anemias from any 
cause and may be related to the red blood-cells as 1 to 10. They are usually 
normal in chlorosis, increased in severe secondary anemia and decreased in per- 
nicious anemia. In splenomyelogenous leukemia we find a large increase of 
these bodies, while in the lymphatic leukemia these cells are diminished. They 
are increased in chronic diseases associated with cachexia and malnutrition, 
being more marked in cancer, nephritis and tuberculosis 1 than in anemia, due to 
other causes. The more acute, more severe, more threatening the disease, the 
fewer the number of platelets 2 so that we have a direct relationship between 
severity of disease and number of these bodies. The method of counting 
these cells has been given in a previous section to which the reader is referred. 
Just exactly what significance is to be attached to their increase or decrease 
is uncertain, but it must be remembered that there is a certain relationship 
between their number and the severity of general conditions. 

(d). Staining Properties. 

These bodies stain very much like nuclear material with a basic stain, 
but also take the acid dyes under certain conditions. With the Wright stain 
these cells are seen as distinctly blue bodies grouped in numbers from 1 to 10 
and seem to be composed of nucleus and protoplasm. The apparent nucleus 
consists of rows of blue or reddish dots occasionally arranged in spherical 
masses, while the indefinite poorly-defined protoplasm-like substance seems 

1 See Webb, Gilbert and Havens, Arch. Int. Med., 1914, XIV, 743; Webb and Gilbert, 
Jour. Am. Med. Assn., 1914, LXIII, 1098. 

2 See Duke, Arch. Int. Med., 1913, XI, 100; also, Port and Akiyama, Deutsch. Arch. f. 
klin. Med., 1912, CVI, 362; Nobecourt and Maillet, Bull. Soc. de Pediat., 1914, XVI, 285; 
Weltmann, Wien. klin. Wchnschr., 1914, XXVII, 1013; Dimond, Jour. Path, and Bacteriol., 
191 5, XIX, 508. 



THE BLOOD. 525 

swollen to almost the size of a red corpuscle. As a rule, however, these bodies 
appear as grouped bluish masses of indefinite structure and outline. 

0). Function. 
The function of these cells is very indefinite. It is possible that they 
have much to do with the formation of fibrin and may be the source of the 
so-called fibrin ferment, thrombogen. 1 If we are to regard these bodies as 
derived from the leucocytes this function of fibrin formation is acceptable, 
but if they are to be considered as derivatives of red cells or as true in- 
dependent bodies such an hypothesis is untenable. No facts of any clinical 
value have so far been forthcoming from the study of the blood plates, and 
it may be possible that they are really artefacts as Lowit has claimed. 

(8). Hemoconien. 

By this term we have reference to the presence in normal blood of very 
fine granules which are actively motile, not truly ameboid, but with motion more 
of the Brownian type. These granules, also called blood-dust by Miiller, are 
small, round, colorless granules which vary in size from very fine dust-like 
particles to some as large as 1 micron in diameter. Their exact chemical 
nature is uncertain as they do not show, according to Miiller, either the reactions 
of fat or of albumin. The general idea prevails that these bodies are the ex- 
truded granules of the leucocytes, as they resemble in size and in staining qualities 
those of various leucocytic types. The number of these granules is uncertain, 
but apparently the relation of these granules to the red cells, as shown by 
the ultra condenser, is about 50 to 1. 

Although no clinical significance is known to attach to these granules, the 
writer has observed marked variations in their number in various pathological 
conditions, but cannot at present draw any conclusions from such observations. 

(9). Morphology of the Blood-forming Organs. 

As the bone-marrow is of such importance in the production of both 
red and white cells, it would seem advisable briefly to discuss the histology 
of this tissue. In it we find practically every cell which occurs in the blood 
both in normal and abnormal conditions, and also many transitional forms 
between various groups of cells. The following brief outline of the histology 
of the bone-marrow is taken from a valuable paper of Dickson. 2 
Varieties of Bone-marrow. 

(1). Primitive or Embryonic Marrow. 

A delicate interlacing network of mucoid cells, which later in the process 
of development go to form the connective-tissue framework or adenoid reticu- 
lum of the tissue. 

(2). Red "Lymphoid" or Formative Bone-marrow Proper. 

This is found in the adult in the short and flat bones, sternum, ribs and 
vertebrae, and to a varying extent in the ends of the long bones. This is the 

1 Bayne- Jones (Am. Jour. Physiol., 1912, XXX, 74) has shown that the platelets aid in the 
clotting of blood in two ways: (1) by setting free prothrombin, which is later activated to 
thrombin, and (2) by liberating a thromboplastic substance which neutralizes the anti-thrombin 
normally present in the blood. 

2 Jour. Path, and Bacterid., 1907, XII, 136. 



526 DIAGNOSTIC METHODS. 

most important variety as in it are found the red and the majority of the white 
cells of the bloo'd, and, according as one or the other of these series of cells 
predominates, the type of marrow may be classified as erythroblastic or leuco- 
blastic, a varying admixture of these two types being practically always found in 
any given case. 

(3). Fatty or Yellow Marrow. 

This is found mostly, as age advances, in the central part of the long bones 
and is formed by a process of physiological transformation or degradation of 
the connective-tissue elements, together with the gradual disappearance of 
most of the blood-forming cells of the red marrow. 

(4). Fibroid Marrow. 

This is found in old persons, especially if debilitated by long-standing 
disease, and is characterized by the proliferation of the connective-tissue ele- 
ments and by the progressive sclerosis of the marrow, followed by the gradual 
disappearance of the hemopoietic cells of the tissue. 

(5). Gelatinous Marrow. 

This is essentially a retrogressive change in the tissue and is in no way 
identical with that type already described as primitive or embryonic marrow. 
This change has been described by a previous writer as a chronic condition 
only, but has been frequently found by Dickson as an acute change in many 
of the acute infectious fevers and allied diseases. 

Cytology of the Bone-marrow. 

In this discussion little more will be taken up than a brief enumeration 
of the various varieties of cells, as the more important ones of these have been 
treated of in other sections. 

(7). Blood-forming Cells. 
(A). Leucocyte Series. 

(a). Nongranular Cells with Basophile Protoplasm. 

(1). Large. 
(2). Small. 

(a). Cells similar to but smaller than the large variety. 
(0). Cells identical in appearance and in staining reactions with 
the small lymphocyte of the blood. Of these there are also 
probably two varieties. 

(b). Granular Cells. 

(1). Neutrophile. 

(a). Myelocytes with large rounded or oval nuclei. These have 
been definitely traced by Dickson to nongranular or hyaline 
cells in which the granules may be seen gradually develop- 
ing. There are two types known as the larger and the 
smaller neutrophile myelocyte. 

(/?). Intermediate cells with indented or horse-shoe-shaped nuclei. 
These are developed from the myelocytes (a) and in turn go 
to form 



THE BLOOD. 527 

(?). Polymorphonuclear cells or adult leucocytes which pass out 
into the blood stream. 
(2). Eosinophile. 
The same types of these cells with eosinophile granulations are found in the 
marrow, as have been previously tabulated under the heading of neutrophile cells. 
(3). Basophile. 
(a). Mast cells. 

The three types above discussed are also observed in the 
basophile mast cells of the bone-marrow. 
(£). Cells resembling the eosinophile myelocytes but with granula- 
tions staining with the basic dye. These cells are probably 
altered eosinophiles. 

(B). Hemoglobin -holding Series. 

(1). Normoblasts Normocytes or ordinary red corpuscles. 

tvt M 1 Normoblasts,. . . normocytes. 

'" 8 \ Megalocytes (entirely pathological in the adult). 

(//). Giant Cells. 

(1). Mononucleated or megakaryocytes. 
(2). Multinucleated or polykaryocytes. 

(///). Cells of Connective-tissue Type. 
(1). Fat cells. 

(2). Cells of the reticulum. 
(3). Various forms of phagocytic cells. 
(4). Ordinary connective tissue cells. 

(IV). Endothelial Cells. 

(1). Found in their normal position in the vessel wall. 

(2). Found proliferating and taking on phagocytic functions. 

Reactions of the Bone-marrow in Disease. 

Many of these reactions are connected with the production of the so- 
called inflammatory leucocytosis and take place with great rapidity. Other 
varieties of change are intimately concerned with the production of the hemoglo- 
bin-holding series of cells. These reactions may, according to the type of cell 
involved, be summarized as follows: 
(7). Leucoblastic. 

(1). Neutrophile. (3). Basophile. 

(2). Eosinophile. (4). Hyaline or non-granular. 

(II). Erythroblastic. 

(1). Normoblastic. (2). Megaloblastic. 

The above brief outline will show the reader that we have in the marrow 
all possible types both of red and of white cells and that any variation in the 
normal activity of the marrow will result in the overloading of the blood with 
cells of a particular type depending upon the kind and extent of the affection. 

The histology of the other blood-forming organs, as the liver, spleen, 
and hemolymph nodes may be found in any text-book on histology. 



528 DIAGNOSTIC METHODS. 

IV. Pathology of the Blood. 
(I). Special Pathology. 

Under the head of the special pathology of the blood we have to consider 
the conditions which are manifested directly by changes in the composition 
and cellular structure of this tissue. In very few of the blood diseases proper 
is the blood picture so characteristic that a definite diagnosis is always possible, 
but in a few of them certain changes are more frequently found and more often 
lead to a presumptive diagnosis. The pathological conditions in the blood 
may be considered either primary or secondary, but it should be remembered 
that severe secondary changes may so closely simulate those found in primary 
conditions that differentiation is almost impossible. It is probably true that 
all pathological changes of the blood are really secondary, but in a certain 
number of these states the etiologic factors are so obscure that we can do no 
more than interpret the blood findings as primary conditions. 
(/I). Anemia. 

This is a condition characterized by a deterioration, both qualitatively and 
quantitatively, in one or in all of the blood constituents. It is usually char- 
acterized by a diminution in the percentage of hemoglobin (oligochromemia) 
and by a decrease in the number of red cells (oligocythemia), but we should 
regard as essential factors in the anemic condition a reduction in the total 
volume of blood (oligemia) as well as a reduction in the amount of protein 
(hypalbuminosis). As much more attention has been paid to the first two 
factors than to the latter ones, anemia has come to mean a reduction in the 
amount of hemoglobin with a more or less extensive reduction in the number 
of red cells. Associated with these conditions we have, in the severer types 
of anemia, variations in form, size, and structure of the red cells as well as 
definite changes in the relationship of the different white cells. In the study 
of the anemic conditions we must differentiate the primary from the secondary 
form, by this we mean a differentiation between those forms which have no 
demonstrable cause from those types whose etiology is more or less secondary 
to other pathologic conditions. 

Primary Anemias. 

(1). Simple Primary Anemia. 

This form which has no demonstrable cause is difficultly separable from 

the secondary form as well as from certain other primary forms, such as primary 

pernicious anemia. It will probably be shown to be a true secondary anemia 

and must be sharply differentiated from the pernicious type from the point of 

prognosis. These cases are only recognizable when they are typical in form 

and are frequently not amenable to any form of treatment which we may 

institute. The rule, however, is that these forms of anemia yield more or less 

promptly to proper dietetic and therapeutic treatment. It is probable that 

the question of prognosis in these cases must depend largely upon the amount 

of degeneration of the red cells which occurs. 1 In these cases we find that the 

1 See Pollitzer (Ztschr. f. klin. Med., 1912, LXXV, 367) for a discussion of the types of 
regeneration and degeneration in anemia; also, Wichern and Piotrowski, Deutsch. Arch. f. 
klin. Med., 1912, CVI, 533; Pribram, Deutsch. Arch. f. klin. Med., 1914, CXVI, 535. 



PLATE XXIII. 




Chlorotic Anemia. (Wright's Stain.) 



THE BLOOD. 529 

diminution in the number of red cells is usually parallel to the reduction in 
the amount of hemoglobin, so that a high color index will usually obtain. 
More or less degeneration, as evidenced by the appearance of poikilocytes, 
normoblasts, Maragliano's polychromatophilia, etc., will be observed depend- 
ing upon the severity of the case. The leucocytes are usually normal in number 
and in differential relations, while the blood plates are usually increased. 
The changes in the plasma in this primary anemia are not characteristic, 
although we do observe a diminution in the specific gravity which runs parallel 
to the oligochromemia. 

(2). Chlorosis. 

Chlorosis is a primary anemia occurring almost exclusively in girls about 
the age of puberty and characterized by a marked reduction in the amount 
of hemoglobin and a slight change in the number of red cells. Clinically, this 
state is evidenced by the appearance of a wax-like changing into a greenish 
tone of the skin and a sky-blue coloration of the cornea. Some cases may show 
a variety of colors of the skin. Certain changes are observed in the digestive 
and generative organs and certain general abnormalities are seen which are 
due to lessened production of blood-cells and to diminished oxidative and 
fermentative powers of the system. This form of primary anemia differs 
from all other forms in the absence of blood degeneration, as very rarely marked 
degenerative signs appear in the blood picture. The blood finding is not 
absolutely characteristic for this clinical entity, as it is simulated by many 
anemias of the secondary type. Clinically, this disease is so sharp that a diag- 
nosis is often possible without a blood examination. 

The chief characteristics of the blood in this condition are: (1) Reduction 
in the hemoglobin. This may run as low as 20 per cent., giving a color index 
of 0.5. Secondary anemias rarely reach such a low level. (2) Variations in 
the number and size of the red cells. The number of red cells is not reduced 
to a very great extent, the average being about 4,000,000, although counts as 
low as 1,000,000 have been reported. When these low counts do occur some 
complication should be suspected. Ordinarily the size of the cell is diminished, 
although we frequently find large "dropsical" cells which are due to absorption 
of fluid from the hypotonic plasma. These latter cells are usually few in num- 
ber, the great majority of cells being smaller than the normal size. Poikilocytes 
and degenerated reds rarely occur except in the severer forms of this disease, 
while chromatophilia is usually regarded as a sign of active regeneration of the 
blood. When nucleated reds occur, which is a rare finding, they are practically 
always of the normoblastic type and rarely appear of the megaloblastic form. 

The leucocytes in this condition are usually normal both in size and 
number and degenerative forms are rarely seen. It has been stated that the 
eosinophile cells are much increased in this condition, but the writer has found 
that their ratio is very rarely above the upper limit of the normal figure for 
these cells. The platelets are usually about normal and are usually large in size. 

In chlorosis we find certain variations in the physical and chemical prop- 
erties of the blood. The specific gravity is usually reduced in proportion to 

34 . 



530 DIAGNOSTIC METHODS. 

the reduction of the hemoglobin and may fall as low as 1028. The plasma is 
usually much diluted so that a condition of oligemia may be considered 
more or less characteristic of chlorosis. Whether or not a hydremia, as 
indirectly manifested by a diminution in the amount of albumin, obtains, is 
debatable. 

Chlorosis belongs to the class of primary anemias and as such has no 
definite etiology. Various conditions such as hypoplasia of the arterial system, 
intestinal autointoxication, disturbances of the nervous system, such as a 
vasomotor neurosis, have been advanced to explain this condition, but none 
of them are tenable in all cases. A great importance must attach to hygienic 
conditions, poor food, and mental depression, especially about the age of 
puberty, as this disease is usually apparent under these conditions at this time 
of life. A further factor which must necessarily bear upon the etiology of this 
condition is the defective power of absorption of iron compounds. Patients 
afflicted with chlorosis improve rapidly under the administration of iron, but 
not unless the digestive and absorptive powers improve at the same time. 
It does not concern us here as to the dynamics of the absorption and the effect 
of iron, but it should be accepted as an axiom that no therapeutic effects may 
be expected from iron unless the absorptive power is made better. Just 
exactly what the pathological conditions are which are accountable for the 
functional insufficiency of the bone-marrow is uncertain, but it must be re- 
called that gross as well as microscopic pathologic changes are not neces- 
sary to produce functional disturbance in any organ. 

It should be remembered that we may have various types of chlorosis 
which show different prognostic characteristics: (1) Those in which the red 
cells are very slightly reduced (about 4,000,000), a marked diminution in the 
amount of hemoglobin, a low color index, and no change in the size and shape 
of the cell. Such cases usually recover promptly without showing any relapse. 
(2) Cases in which the red cells are below 4,000,000, which show a marked 
diminution in the amount of hemoglobin with a very low color index and which 
give very slight evidences of degenerative changes in the red cells. These cases 
are usually characterized by marked prostration, but usually recover more or 
less promptly although relapses frequently occur. (3) Cases in which the red 
cells are reduced as low as 2,500,000, a reduction in the hemoglobin with a very 
low color index, marked changes in the shape and size of the red cells. These 
cases respond slowly to treatment and have a bad prognosis (Ewing). 

As these cases of chlorosis convalesce, we observe an increase in the 
number of red cells to a point somewhat above the normal and a later increase 
in the hemoglobin content of each individual cell. These changes are usually 
evident in from eight to ten days after institution of treatment, but as a rule 
a much longer time is necessary for any marked change to be observed in the 
number of the cells or in their hemoglobin content. The changes in the plasma 
are usually the first to appear and should be considered essential for the proper 
regeneration of the blood in chlorotic conditions. 

This disease belongs in the group of curable conditions and usually has a 
good prognosis, but the susceptibility to intercurrent acute diseases during 



PLATE XXIV. 




Blood in Pernicious Anemia. (Wright's Stain.) 



THE BLOOD. 53 I 

this period is much increased so that we should always bear in mind the proba- 
bility of complications arising in the convalescent period. 

(3). Progressive Pernicious Anemia (Biermer's Anemia). 
This term is applied to a form of severe anemia which, in spite of all 
treatment, progresses steadily toward death. It results from defective hema- 
togenesis and excessive hematolysis and is characterized by definite changes, 
both numerically and morphologically, in the red cells and by characteristic 
changes in the bone-marrow (Ewing). This condition has frequently been 
described as the result of infection with certain intestinal parasites so that it 
cannot in all cases be considered a truly primary disease, although the larger 
majority of cases show no known etiology. 1 According to Herter, certain 
anerobic bacteria, found in the large intestine, produce a substance of marked 
hemolytic power, which penetrates the intestinal wall and enters the portal 
circulation. 2 In some cases of idiopathic purpura hemorrhagica the blood 
picture is that of pernicious anemia, but may be distinguished by the absence 
of megaloblastic change and by the prominence of hemorrhage. The blood 
picture in this disease is not absolutely characteristic, as certain forms of 
secondary anemia may show similar findings. The chief characteristics of 
the blood in primary pernicious anemia, as also in those severe types of 
secondary anemia which simulate this form, are (1) signs of rapid blood de- 
struction, such as degenerated reds, endoglobular degenerations, polychroma- 
tophilia, increased iron compounds in the serum and corpuscles, an increase 
of iron in the liver and spleen; (2) poikilocytosis; (3) a high color index resulting 
from a marked diminution in the number of red cells and a correspondingly 
less degree of diminution in the amount of hemoglobin; (4) megaloblastic blood 
formation. This latter indicates a direct reversion to the embryonal type 
of blood formation in which the presence of megaloblasts as direct pre- 
cursors of megalocytes is observed. 3 The red blood-cells in this condition 
are few in number being reduced to as low as 1,000,000 cells, counts of 
500,000 having been observed without the patient suffering any marked in- 
convenience. Naegeli reports a case showing 138,000 reds. This fact 
should be taken as evidence that the oligocythemia in pernicious anemia is 
not alone accountable for the symptomatology. This count may remain 
stationary, may show slight decrease, but usually progresses slowly until death 
ensues. The average diameter of the red cells is somewhat increased in 
pernicious anemia. While many of them may be normal in size and many 
very small, the cells average from 4 to 13 microns in diameter. A pernicious 
anemia is a distinct large cell anemia. A macrocytosis is much more character- 
istic of this disease than of any other, 70 per cent, of the cells in these cases being 
of this type. 4 Microcytosis is rare but may occur to such an extent that the 

1 See Friedstein, Inaug. Dissert., Berlin, 1912; Liidke and Fejes, Deutsch. Arch. f. klin. 
Med., 1913, CIX, 433; also Pilcher, Am. Jour. Med. Sc, 1913, CXLVI, 226; Ragosa, Folia 
Haemat, 191 5, XIX, 269. 

2 See Banti, Semaine med., 1913, XXXIII, 313; also, Robertson, Jour. Biol. Chem., 1915, 
XVI, 652. 

3 See Brcsamlen, Deutsch. Arch. f. klin. Med., 1913, CXII, 83; Vogel and McCurdy, 
Arch. Int. Med., 1913, XII, 707; Roth, Ztschr. f. klin. Med. 1914, LXXIX, 266. 

4 See Dianner, Berl. klin. Wchnschr., 1914, LI, 1759. 



532 DIAGNOSTIC METHODS. 

average size of the red cell may be about normal. Poikilocytes are very 
common and often show extreme shapes and are frequently numerous in 
number. Polychromatophilic degeneration is very extensive in this form of 
anemia. 

While both normoblasts and megaloblasts occur in large numbers in per- 
nicious anemia, the megaloblasts usually outnumber the normoblasts. This 
megaloblastic increase may be considered pathognomonic of this anemia, as a 
preponderance of these large nucleated red cells does not occur, except in rare 
cases, in the other varieties of anemia. The hemoglobin may be markedly 
reduced showing values rarely above 50 per cent, and often as low as 10 per 
cent. The color index is always high ; being more frequently above one than 
below. A low index is found, according to Ewing, in the chronic cases, while 
the acute forms are more frequently associated with a high index. If im- 
provement occurs the index is always lowered, an increasing index denoting 
a bad prognosis. 

In this pernicious form of anemia we find the leucocytes practically al- 
ways diminished in number, averaging about 4,000, a condition which almost 
never obtains in a secondary anemia. Their number usually runs parallel to 
that of the red cells, a leucocytosis pointing probably to a complication. As 
the case improves the neutrophile cells increase in number, the low leucocyte 
count being, as a rule, due to their diminution. The percentage of the non- 
granular mononuclear cells varies inversely to that of the granular form. As 
the disease progresses the percentage of the nongranular cells increases, while 
the granular cells are diminished. This disease shows, therefore, a high lym- 
phocyte count which is relative and not real, being due to the diminution in 
number of the polymorphonuclear cells. The lymphocytes may constitute 
as high as 50 per cent, of the leucocytes, the eosinophiles may reach as high 
as 10 per cent., the myelocytes 2 per cent., while the mast cells may run as high 
as 3 per cent. Degenerations of all kinds are observed in the leucocytes, but 
nothing characteristic of pernicious anemia is found in the white cells. 1 

The blood-platelets are largely decreased and may be as low as one- 
twenty-fifth of the normal number. Von Limbeck and Sahli claim that these 
cells are increased in number, but the usual finding is one of diminution. 

(4). Splenic Anemia. 
This is a form of chronic anemia characterized by idiopathic enlargement 
of the spleen without any involvement of the lymph nodes. A large number 
of conditions may be responsible for the anemia of the splenic type, so that a 
direct etiologic factor should be looked for in all cases. Among the conditions 
which may give rise to this type of anemia, we find gummata of the spleen, 
large round-cell sarcoma of the spleen, chronic splenitis of the malarial type, 

1 See Nicol, Deutsch. Arch. f. klin. Med., 1913,0x1,417; Sisto, Policlinico, 1913, XX, 509; 
Ibid., 1914, XXI, 34, 91 and 115; Wolff, Deutsch. Med. Wchnschr., 1914, XL, 643; Cederberg, 
Berl. klin. Wchnschr., 1914, LI, 585; Mikhailoff, Russk. Vrach., 1914, XIII, 837; Cavaglieri, 
Gazz. d. osp., 1914, XXXV, 1169; Schmidt, Am. Jcur. Med. Sc, 1914, CXLVIII, 313; Brig^s, 
Ibid., 413; Momtt, Ibid., 817; Krumbhaar, Ibid., 1915, CL, 227; Drinker and Hurwitz, Arch. 
Int. Med., 1915, XV, 733. 



THE BLOOD. 533 

and splenomegaly associated with cirrhosis of the liver. This latter condition 
is known as Banti's disease and its etiology is uncertain. 1 

While it is true that the splenic lesions do not differ essentially from 
certain stages of the lesions in ordinary pseudoleukemia, yet we do not find 
in this condition any involvement of the lymph-glands, although the spleen 
may be enormously enlarged. The blood picture in this condition is character- 
ized by a relatively high red cell count, a marked reduction in the percentage 
of hemoglobin, and a consequent low color index. The white cells are rarely 
increased, a leucopenia being rather the rule. When we do find an increase 
in the number of white cells a relative lymphocytosis occurs usually associated 
with an increase in the number of basophiles. Poikilocytes and nucleated red 
cells are very uncommon while degeneration may occasionally be observed. 
Whether or not we have an increase or a decrease in the number of 
leucocytes, a relative lymphocytosis associated with enlarged spleen and no 
involvement of the lymph nodes must be considered characteristic of splenic 
anemia. 

In Banti's disease we find an enormous increase in the size of the spleen 
and associated with this an extensive cirrhosis of the liver. Along with 
these factors we find a high-grade toxogenic protein decomposition, which is 
associated with very high values for the total nitrogen of the urine and of the 
output of purin bases. The number of erythrocytes diminishes corresponding 
to the degree of anemia, while the hemoglobin percentage is more markedly 
reduced. The leucocytes are either normal or more frequently diminished 
in number, a relative lymphocytosis existing as in the pure type of splenic 
anemia. 2 

(5). Anemia Infantum Pseudoleukemica. 

Von Jaksch has described a rare form of anemia seen in children which 
is characterized by enlargement of the spleen, liver, and lymph nodes. The 
most striking points in this condition are the great diminution in the number 
of red cells, one case showing only 820,000; numerous nucleated red cells; 
diminution of hemoglobin; the leucocytes always increased in number, being 
from 20,000 to 50,000, as a rule, and displaying a remarkable variety of form and 
frequently attaining to unusual size. The morphological changes in the blood 
resemble both those seen in leukemia and in pernicious anemia, the disease 
passing either into one or the other of the previously mentioned conditions. 
The blood findings are not alone sufficient to warrant a diagnosis of infantile 
pseudoleukemia, but are significant when taken in conjunction with the clinical 
findings. 

1 See Banti, Berlin, klin. Wchnschr., 1911, XLVIII, 2328; Stein, Am. Jour. Med. Sc, 1912, 
CXLIV, 856; also, Umber, Munch, med. Wchnschr., 1912, LIX, 1478; Lacouture, Duperie, 
and Charbonnel, Jour, de med. de Bordeaux, 1913, LXXXIV, 805; Caronia, Pediatria, 1914, 
XXII, 752; Sailer, Penn. Med. Jour., 1914, XVIII, 91; Krull, Mitt. a. d. Grenzgeb. d. Med. u. 
Chir., 1914, XXVIII, 718. 

2 Yates, Bunting and Kristjanson (Jour. Am. Med. Assn., 1914, LXIII, 2225) report the 
isolation of pure cultures of Bacillus hodgkini (see Chap. XI) in two cases of Banti's disease. 
They believe there is an etiologic relationship here. See, also, Gibbons (Quart. Jour. Med., 
1914, VII, 153), who reports the presence of a streptothrix in six cases. 



534 DIAGNOSTIC METHODS. 

(6). Leukanemia. 

This condition has been classed by some with the leukemias, as the blood 
findings are occasionally more prominent among the leucocytes, while by others 
it is classed with pernicious anemia owing to the frequent changes in the red 
cells. Von Leube considers this anemia a mixed form of pernicious anemia 
and of leukemia. Luce 1 regards this as a symptom of many conditions rather 
than an independent blood disease. In the majority of these cases the changes 
are more evident in the red cells, marked diminution in number (as low as 
200,000 per cmm.) associated with extensive destruction of red cells with all 
irregular and unusual types of these cells being observed. The diminution in 
the amount of hemoglobin is great, but the color index is usually high just as we 
find it in pernicious anemia. The white cells show an extensive disturbance 
in the neutrophile and eosinophile blood picture with a large increase in the 
number of large lymphocytes. The number of white cells is usually increased, 
but not to as great an extent as in leukemia. The changes in the red cells 
and in the hemoglobin usually precede those in the white cells, so that the 
early stages may show us a typical picture of pernicious anemia, while later 
examinations may lead to the diagnosis of the mixed condition. 

(7). Aplastic Anemia. 

Ehrlich has reported a rapidly progressing anemia, accompanied by 
hemorrhages into the mucous membranes, associated with hyperplasia of 
the bone-marrow, and not showing the ordinary changes in the blood which 
are supposed to accompany pernicious anemia. This type has been called 
aplastic anemia and has not been frequently reported. 

The red cells are usually markedly reduced in number, being as low as 
790,000 in a case reported by Wood. The hemoglobin may fall as low as 
1 1 per cent, as reported by Muir, while the leucocytes are usually normal in 
number but showing much reduced percentages of the polynuclear neutrophiles. 
In this condition we find an enormous increase in the number of lymphocytes, 
the percentage being in Lipowski's case 93, the remaining 7 per cent, being 
neutrophiles. No nucleated reds have been found in the blood and only a 
very few in the marrow itself, which is fatty, almost white and contains few 
neutrophiles and no eosinophiles. 2 

Secondary Anemia. 

By secondary anemia we mean one in which etiological factors seem 
sufficient to explain the variations observed in the blood. The principal 
variation seems to be more directly observed in the reduction of the hemoglobin, 
although the number of red cells is coincidently reduced, but not to the same 
degree as is the hemoglobin. In the mild cases the color of the blood is but 
slightly paler than normal, but in secondary anemia of a severe type the color 
may resemble the watery drop observed in cases of typical pernicious anemia. 

Cabot suggests a classification of the secondary anemias as follows: 

1 Deutsch. Archiv. f. klin. Med., 1900, LXXVII, 215. See, also, Martelli, Virchow's Arch., 
1914, CCXVI, 224; Gazz. internat. di med., 1914, XVII, 553 and 577. 

2 See Hirschfeld, Folia Haemat., 1911, XII, 347; Musser, Arch. Int. Med., 1914, XTV, 275; 
Kleinschmidt, Jahrb. f. Kinderhkde., 1915, LXXXI, 1. 



FLATE XXV. 




Blood in Leukanemia. (Wright's Staix.) 



THE BLOOD. 535 

(i) Mild cases, showing a normal count of red cells, but having the hemoglobin 
diminished. (2) Moderate cases in which the count is normal but the cells 
shoW signs of moderate degeneration, abnormal staining qualities, and a 
diminished tendency to rouleaux formation. (3) Severe cases, in which the 
count is not much reduced but in which the hemoglobin is very much lessened 
and the cells show marked qualitative and quantitative changes. (4) Very 
severe cases with a slightly lessened blood count, a marked diminution of the 
hemoglobin, and evidences of degeneration ahd destruction of the cells as well 
as evidence of regeneration. In some of the severer types of secondary anemia 
the blood picture may so closely resemble that of pernicious anemia that a 
diagnosis is possible only through the careful investigation of the etiology 
of the condition. 

Blood Picture. 

The general points observed in secondary anemia are as follows: A 
variable decrease in the amount of hemoglobin, a reduction in the number 
of red cells less in degree than the diminution of hemoglobin and a subnormal 
color index, which rarely reaches the low grade shown in chlorosis. The 
lowest color index is seen in those secondary anemias following cancer, severe 
hemorrhage, and gangrenous processes. The specific gravity of the blood is 
reduced corresponding to the degree of reduction of hemoglobin. The rapidity 
of coagulation is increased depending upon the grade of oligochromemia and 
of oligocythemia. The reduction in the number of red cells may be very 
marked as, for instance, in a case of von Limbeck the number was 306,000. 
The red cells show a lack of hemoglobin, frequently appearing as the pessary 
forms. Polychromatophilia is quite common, but bears no direct relation to 
the hemoglobin content of the cell. Only in the severer cases do we find 
poikilocytes, although anisocytes, especially microcytes, are frequently present, 
the larger "dropsical" cells being less common than in chlorosis. Nucleated 
erythrocytes are frequently seen in some cases, while in others even of severer 
grade of anemia they are absent. These nucleated reds are, as a rule, of 
the normoblastic variety, megaloblasts being exceedingly rare. 

The leucocytes vary in number depending on the cause of the anemia, 
from a leucopenia, which is rare, to a leukemic condition. The increase in the 
number of leucocytes is more frequently in the polynuclear neutrophils, the 
lymphocytes being rarely if ever increased, while the eosinophiles, although 
not increased, are usually at the upper limit of the normal value for these cells. 
In some cases of the severe chronic types of secondary anemia we may find a 
lymphocytosis, but this is rare. The blood-platelets are usually increased in 
number, in some cases being two and one-half times the normal values. 

(1). Acute Hemorrhage. 

The character of the anemia following an acute hemorrhage will depend 
upon the type of the hemorrhage, that is whether the loss of blood occurred 
suddenly and at one period, or whether slowly and at intervals. 1 The loss of 

1 See Milne, Jour. Exper. Med., 1912, XVI, 325; Deutsch. Arch. f. klin. Med., 1913, CIX, 
401. 



536 DIAGNOSTIC METHODS. 

one-half of the total volume of blood at one time is usually fatal, asPanum has 
shown. If the loss be less following one large hemorrhage, regeneration takes 
place in from five to thirty days, depending upon the amount of blood lost. 
Regeneration is quickest in men between the ages of 20 and 40, slower in 
women, and slowest in children. 

Immediately following a hemorrhage the blood picture will be normal 
qualitatively as there has been no time for the morphologic changes to take 
place. Shortly following the hemorrhage lymph pours into the blood to re- 
store the volume and maintain the pressure, so that the blood count and the 
hemoglobin diminish usually to the same degree. As the formation of new 
cells goes on the color index decreases, as the new cells are, as a rule, deficient 
in hemoglobin. These cells are more or less easily degenerated, showing varia- 
tion both in their shape and staining qualities. The number of cells reaches 
the normal much sooner than does the percentage of hemoglobin, so that we 
may find for weeks evidences of marked anemia. 

The most frequent causes of acute hemorrhagic anemia are traumatism, 
ectopic pregnancy, abortion, gastric and duodenal ulcers, uterine tumors, 
pulmonary tuberculosis, and hemorrhagic pancreatitis. 

(2). Chronic Hemorrhage. 

By a chronic hemorrhage we mean one in which repeated hemorrhages 
follow one another so closely that the blood has no time to regenerate before 
a second loss of blood occurs. This rules out of consideration those cases in 
which repeated hemorrhages occur but at sufficiently long intervals to permit 
of regeneration of the blood. This latter condition, although a chronic hemor- 
rhage, gives the picture described under Acute Hemorrhage. 

In the chronic hemorrhage we find the red cells markedly reduced, the 
hemoglobin very much diminished and usually a marked leucocytosis. The 
red cells are usually small and pale, show a low color index, and usually few 
nucleated forms, although the picture may rarely assume the pernicious type. 
Regeneration in this form of anemia is slow, as the blood-forming organs seem 
to lose their power of regenerating the blood after repeated hemorrhages. 

The most frequent causes of chronic hemorrhage are scurvy, epistaxis, 
hemorrhoids, intestinal ulcers, gastric and other carcinomata, and intestinal 
parasites. 

(3). Inanition. 
In the discussion of the anemia of inanition, it must be remembered that 
other factors than starvation are necessary in its causation. That starvation 
alone will not cause anemia is shown from the examination of the blood of 
Cetti who fasted ten days. His blood showed about 6,000,000 red cells, a 
small diminution in the percentage of hemoglobin, and a leucocyte count of 
4,200. Although the changes in the cells and the pigment of the blood are not 
marked following starvation, it is true that we have a loss of albumin of the 
plasma and a diminution in the total volume of blood. This is taken as 
evidence by Grawitz that a true anemia occurs. Such changes are not striking 
if the days of fasting are alternated with days of slight nourishment. It is not 



THE BLOOD. 537 

so much the quantity of food as the quality which is of importance in bringing 
about the anemic conditions. As is well known, the foods containing iron are 
the principal sources of the hemoglobin of the blood, and these are frequently, 
owing to disturbed gastric and intestinal functions, poorly digested and assimi- 
lated. Although the amount of iron contained in the ordinary food is sufficient 
under the best conditions to maintain the hemoglobin content of the blood, 
yet the methods of food preparation, as well as the abnormal methods of rapid 
eating, are important factors in the poor assimilation of the iron of the food. 
The lack of sunlight as well as impure air are contributory factors in 
causing anemia through their influence upon the general body functions. No 
tissue, whether animal or plant, can flourish in air which does not have sufficient 
oxygen to support the combustion processes of the system. This statement 
needs no retraction in the case of anerobic bacteria, as it has been definitely 
shown that these organisms obtain the oxygen necessary for their develop- 
ment from the culture media upon which they grow, although they are inca- 
pable of developing in an atmosphere of pure oxygen. Overwork, especially 
when associated with worry, has great influence upon all of the functions of 
the system. For this reason overwork has been credited with the power of 
producing anemia as well as many other serious systemic disturbances. How- 
ever, it is rare to find an authentic case of anemia which can be traced directly 
to overwork without the mental influence of worry aud the coincident nervous 
strain from this latter cause. 1 

(4). Intestinal Parasites. 
The anemia caused by intestinal parasites may be of such a severe grade 
as to resemble very closely the type of pernicious anemia. In many cases it 
is an impossibility to make a differential diagnosis between these types with- 
out the finding of an intestinal parasite which will clear up the diagnosis. In 
these cases the blood picture returns more or less quickly to normal after 
removal of the parasite in question, while in the pernicious anemia of un- 
known origin the progress of the disease is always toward a fatal termination. 
It is probable that the cause of the severe secondary anemia due to the presence 
of the intestinal parasite is a result of the toxic condition set up by the absorp- 
tion of the hemolytic toxins elaborated by the parasite. A very severe anemia 
of the secondary type is frequently seen as a result of decomposition of the in- 
testinal contents and in cases of chronic constipation in which the direct toxic 
agent is at present unknown. The most common intestinal parasites causing 
the severe types of secondary anemia are (1) uncinaria duodenale, (2) strongy- 
loides intestinalis, and (3) bothriocephalus latus. The first of these causes an 
anemia which is very closely related to that shown by miners and tunnel diggers, 
and seems to be much more prevalent in the southern part of the United 
States, although it occurs in many different countries. The blood count may 
fall below one million red cells and the hemoglobin may be as low as 15 per 
cent., while all varieties of degenerative changes may be seen in the erythrocytes. 
In the anemia following infection with the bothriocephalus we find very marked 
1 See Ash, Arch. Int. Med., 1914, XIV, 8. 



538 DIAGNOSTIC METHODS. 

similarity with the primary pernicious type. One-half to two-thirds of the 
nucleated reds in this variety may be of the megaloblastic type and yet may 
disappear within two to three weeks after the worm has been expelled. It is 
uncommon to find in secondary anemias caused by the parasites above men- 
tioned any marked eosinophilia, which is so common in cases of infection, with 
many of the other forms of intestinal parasites. The anemia of the severer type 
seems to prevent a chemotaxis toward eosinophile cells. 

(5). Fever. 
It is still very much of a question whether the blood in febrile cases shows 
the characteristics of a secondary anemia, as the result of the temperature 
increase. It has been shown that increased temperature, in itself, does not 
always produce anemia, although we do have marked destruction of the red 
cells and a coincident loss in the amount of hemoglobin of the remaining cells. 
So great is the influence of the toxin of the febrile condition that it is highly 
probable that the anemia so frequently observed in febrile cases is due to a 
combination of causes, rather than to a specific effect of the increased tem- 
perature. The 'changes in the white cells in febrile cases are not always of 
the same character, the variations being dependent upon the specific causative 
factor of the fever. As the blood changes in the acute infectious fevers are of 
more or less importance, they will be discussed later under separate headings. 
The anemias which are secondary to both the acute and chronic infections 
are probably directly due to the influences of the toxins upon the blood and 
blood-forming organs. The condition of general nutrition as well as the state 
of digestion, especially in the chronic states, such as tuberculosis, leprosy, and 
syphilis, must be regarded as important factors in the causation of these second- 
ary types. 

(6). Blood Poisons. 

There are a very large number of compounds which produce, when 
taken in toxic doses, very marked changes in the qualitative and quantitative 
composition of the blood. As is well known, iron compounds in therapeutic 
doses increase the amount of hemoglobin in the red cells and also increase 
the number of red cells up to a certain point. Many of the effects which are 
attributed to iron compounds may be due to the improved hygienic and dietetic 
conditions which usually prevail during the administration of these substances. 
Yet the therapeutic results following the administration of iron are such as 
to make it certain that a specific influence of this drug is present in anemic 
conditions, especially of the chlorotic type. 

Many compounds produce a very marked secondary anemia, the most 
important of these being alcohol, opium, lead compounds, cocain, and acetani- 
lid. While others, such as arsenic, nitrobenzol, nitroglycerin, phenacetin, 
and poisonous mushrooms, cause dissolution of the red cells with marked 
hemoglobinemia. The anemia following the use of lead, either in toxic doses 
or after its slow absorption from constant contact with it in the arts, is of great 
practical importance. The causes of this lead anemia are rather complex. 
The lead compounds have a direct action on the red cells and on the blood- 



THE BLOOD. 539 

forming organs as well as upon the gastrointestinal tract and the eliminative 
organs. While the anemia shows no especial characteristics as regards the 
number of red cells and the amount of hemoglobin, yet the peculiar granular 
degeneration and the polychromatophilia are sufficient to differentiate this 
type from most of the other secondary anemias. The basophilic degenerations 
of the reds is more marked in lead anemia than in almost any other condition 
and usually runs parallel to the severity of the clinical symptoms of the case. 
In arsenical poisoning also we occasionally find a slight amount of granular 
degeneration of the red cells, but the hemoglobinemia in this latter condition 
will differentiate it from lead anemia. 

(B). Leukemia. 

Although acute forms occur, leukemia may be regarded as an essentially 
chronic condition which is characterized on the one hand by definite changes 
in the lymphatic and myeloid tissues of the body and, on the other hand, by 
certain peculiar changes in the number and relations of the various cellular 
constituents of the blood. These latter conditions must be regarded as purely 
symptomatic of the preceding states and not as the direct pathological condi- 
tion in themselves. 1 Leukemia has been classed as a primary anemia, although 
the changes in the blood are here more directly related to variations in the 
white cells than to characteristic changes in the red corpuscles; yet we do find 
a diminished red count as well as a diminution in the amount of hemoglobin 
in this condition. It is a disease marked by the constant presence in the 
blood of granular mononuclear or polynuclear cells, or an increase of the non- 
granular cells with round nuclei. While the leucocyte count is almost in- 
variably increased to a marked extent, we find cases in which the number of 
cells is normal, but we find great deviations from the normal relations of 
these white cells. 

While the tendency is becoming more and more general to regard this 
condition as a single entity, manifested by various blood pictures, yet we 
find the cells grouping themselves together in such definite ways that we 
are justified in dividing leukemia into three general types, with transitions 
from one to the other form. 2 These types are (i) spleno myelogenous leukemia 
or "myelemia," (2) lymphatic leukemia or "lymphemia," (3) mixed leukemia. 
Each of these types shows a distinct blood picture which permits of the classi- 
fication of the condition studied. 

(1). Splenomyelogenous Leukemia. 

This condition was formerly subdivided into two distinct types— the true 
splenic and the myeloid leukemia. However, practically none of the cases 

1 It is probable that leukemia, both of the chronic and acute types, is of infectious origin. 
Streptococci and the bacillus hodgkini have both been isolated from these cases. See Steele, 
Boston Med. and Surg. Jour., 1914, CLXX, 123; Simon and Judd, Jour. Am. Med. Assn., 
1915, LXIV, 1630; Wilbur, Ibid., 1915, LXV, 1255. 

2 It is probably true that a classification of these types on purely morphological bases is 
futile. See von Bombard, Ztschr. f. klin. Med., 1914, LXXX, 506; Emden and Rothschild, 
Deutsch. Arch. f. klin. Med., 1914, CXV, 304; Panton, Tidy and Pearson, Quart. Jour. Med., 
1914, VII, 304: Rummo, Rif. Med., 1914, XXX, 897 and 926; Castellino, Ferrata and Rummo, 
Gazz. d. osp., 1 914, XXXV, 1813; Rotky, Zentralbl. f. inn. Med., 1914, XXXV, 953; Martelli, 
Rif. Med., 1914, XXX, 1233, 1271, 1294 and 1324. 



54Q 



DIAGNOSTIC METHODS. 



reported could be definitely classed under either one of these headings, as the 
blood picture was always referable to disturbance in both the spleen and 
marrow. This type of leukemia is characterized by a marked increase in all 
of the granular cells, especially of the neutrophile, eosinophile, and basophile 
types, while the nongranular cells are not so characteristically increased. 

Gross Appearance. 

The gross appearance of the blood is normal even though the leucocytes 
are increased to an enormous extent. In extreme cases it may appear pale 
and opaque and does not flow from a puncture as readily as normal blood. 
In making smears of such blood, the preparations appear granular and are not 
readily spread so that the future examination is rendered somewhat difficult. 

Red Cells. 

As a rule, these cells are diminished in number, but the oligocythemia 
is of a mild degree, the average count being about 3,000,000, although it may run 
as low as 1,500,000. As a general rule, the red cells diminish in proportion 
to the increase in the number of white cells. Occasionally we find cases in 
which an oligocythemia persists with a normal or slightly increased leucocyte 
count. Such a condition might lead to the diagnosis of pernicious anemia, 
unless the differential count was carefully studied. The red cells are usually 
pale and of the chlorotic variety. Very little degeneration of the red cells is 
observed, microcytes and macrocytes are rare, but a few poikilocytes are seen 
in practically all cases. Polychromatophilia is more or less common and cells 
showing basophilic granulations appear with more or less frequency. Normo- 
blasts are very common in this condition, yet their absence does not rule out 
the diagnosis of leukemia. Megaloblasts and gigantoblasts are frequently 
seen and are sometimes many in number, although they rarely if ever exceed 
the normoblasts in number. 

Hemoglobin. 

The hemoglobin is reduced to a somewhat greater extent than is the number 
of red cells, the color index being about 0.6 and the average percentage of 
hemoglobin about 40. 

Leucocytes. 

In this condition we find the leucocytes increased, as a rule, to a very 
marked degree, counts running as high as 750,000 having been seen by the 
writer. Osier gives as* his average for the white count 298,700, while the 
average may vary to a slight extent at different periods of the day. Some of 
the cases show a uniformly high count, others a moderate count and a few 
others a low count of about 100,000 cells. It is this increase in the number 
of white cells which gives the blood its peculiar opacity in this condition and 
may make a diagnosis possible by mere inspection. In some cases of leukemia 
we find a normal count of white cells while in others the count may be similar 
to that of a simple leucocytosis. It is the differential count in combination 
with the large increase in cells which should be considered characteristic, 
rather than a simple increase in itself. 



PLATE XXVI. 




Blood in Spleno-myelogenous Leukemia. (Tri-acid Stain.) 



THE BLOOD. 541 

Differential Count. 
Neutrophile Myelocytes. 

These cells are large mononuclear cells with neutrophile granules. They 
are present in large numbers, averaging about 35 per cent, of all the leucocytes 
present. A diagnosis of leukemia is almost always possible when we have 
such an extreme neutrophile myelocytosis along with an extreme leucocytosis. 
These myelocytes appear in two forms: (1) the large myelocytes of Cornil, 
which may be as large as 30 microns in diameter and have a large, pale, eccen- 
tric nucleus which is poor in chromatin. These cells are seen only in spleno- 
myelogenous leukemia and in some of the secondary leukemias of children 
and must be regarded as practically pathognomonic of this condition. 1 (2) 
Small myelocytes about the size of the normal polynuclear leucocytes with 
a centric, round nucleus staining deeply with the various aniline dyes. We 
find all gradations between these large and small myelocytes, sometimes 
observing a few which are about the size of the red cell. The granulations 
of these cells are sometimes numerous, but may be entirely lacking so that 
they may be indistinguishable from the large lymphocytes unless the pale 
quality of their nuclei is remembered. The degenerative changes in these 
myelocytes are few in number and are limited, as a rule, to the hydropic form 
usually seen in chlorosis. 

Polynuclear Neutrophile Leucocytes. 

These cells are relatively diminished, their average, according to Cabot, 
being about 46 per cent., although an absolute increase is present, amounting 
to as much as 60,000 to 75,000 cells. Marked variations in the size of these 
cells are common, some of them being very large, some very small and no defi- 
nite relation existing between the numbers of the large and small cells. These 
variations in size of the polymorphonuclear cells are rarely if ever seen in 
ordinary leucocytosis. It is very common to find cells with irregularly shaped 
nuclei and with more than one form of granule, which may vary in tint depend- 
ing on the method of fixation. Marked degenerative changes in these cells 
are very common. Thus we find their stickiness is increased, their nuclei 
usually pale and frequently showing karyokinetic figures. All grades of varia- 
tions in the granulations may be observed, the granular cytoplasm being oc- 
casionally replaced by a homogeneous highly refractive material. 

Eosinophiles. 

The eosinophile cells are usually much increased in the splenomyelogenous 
form of leukemia, but their percentage relations to the other leucocytic forms 
are practically normal. Their number may run from 3,000 to 100,000, the 
average absolute number being about 12,000, while their percentage is about 
five. The total number of these cells per cmm. greatly exceeds that found 
in any other condition so that we accept, with Ehrlich, such an increase as 
pathognomonic of splenomyelogenous leukemia. These cells appear in all 
modifications, some of them being very small, while some of them are very 
large. The eosinophile form of myelocyte occurs in large numbers, but never 

x See Klein, Deutsch. med. Wchnschr., 1913, XXXIX, 2513. 



542 DIAGNOSTIC METHODS. 

is as numerous as is the neutrophile myelocyte. We occasionally observe all 
forms of transition between the myelocyte and the eosinophile leucocyte, the 
eosinophile myelocytes occasionally forming the majority of the eosinophile 
cells. The granulations of these cells may be of uniform size and staining 
quality or there may be some basophile granulations among the eosinophiles 
while the granulations themselves may vary greatly in size. Ewing considers 
eosinophile myelocytes with granules of unequal size and density of stain as 
pathognomonic of myelocythemia. 

Basophiles. 

According to Ehrlich, we always find an increase in the number of mast 
cells in leukemia, their absolute increase being in some cases greater than 
that of the eosinophiles and is always proportionately higher. This increase 
is so marked as to constitute a very reliable diagnostic feature of the blood. 
The number of basophiles may run as high as 140,000 (Taylor), while the 
percentage may vary from 5 to 47. 

Lymphocytes. 

The number and proportions of lymphocytes in the splenomyelogenous 
leukemia vary in different cases and at different times in the same case. As 
a rule, their percentage is reduced averaging about 10, while an absolute in- 
crease is usually present, this increase having no uniform relationship to 
the stage or character of the disease. These cells vary much in size, the 
large cells usually outnumbering the small ones. Large mononuclear cells 
with very faint cytoreticulum and vesicular nucleus occur in large numbers 
in this form of leukemia and seem to have no special significance, although 
they may be mistaken for the large lymphocyte or for the myelocyte. Degen- 
erative changes are observed in both the small and large lymphocytes in leu- 
kemia; thus the nuclei of the small cells may become incurved and bilobed or 
even trilobed, while the cell body remains basophilic (Rieder). 

Points in Diagnosis. 

An excessive leucocytosis, with a large proportion of neutrophile mye- 
locytes, the presence of a large number of eosinophile myelocytes and of 
basophile cells, the presence of atypical cells, both of the mononuclear and 
polynuclear variety, and large numbers of nucleated red cells are the chief 
characteristics. Any one of these points may fail for a time, but will usually 
be evident at some stage of the disease. The large size of the myelocyte 
is much more characteristic than the mere presence of these cells, so that 
at should confine our diagnosis to cases which show irregularity in size, staining 
qualities, and degenerative reactions of these cells rather than to those show- 
ing merely an increase. The presence of the large number of eosinophiles, 
especially those showing granules of irregular size and staining qualities, is 
a very important point to be remembered in the diagnosis of this condition. 1 

1 See Dieballa and Entz., Folia Haemat, 1913, XV, 59; also, Ghon and Roman, Ibid., 72; 
and Milne, Jour. Am. Med. Assn., 1913, LX, 822. Goodall (Boston Med. and Surg. Jour., 
1914, CLXX, 789) discusses the nitrogenous metabolism in this condition. 



PLATE XXVII. 




Lymphatic Leukemia. (Tri-acid Stain.) 



THE BLOOD. 543 

(2). Lymphatic Leukemia (Lymphemia). 

In this form of leukemia we observe a marked increase in the number 
of mononuclear nongranular cells in distinction from the previous form of 
leukemia in which the increase is rather in the number of the granular types. 
While a variety of the mononuclear nongranular cells are present, there is 
usually observed a predominance of one particular form and size, in some 
cases the small mononuclear cell with a narrow ragged rim of protoplasm, in 
others the cells of the large lymphocyte type, and in others large cells whose 
protoplasm is basophilic or in some cases distinctly acidophilic. 1 

Red Blood-cells. 

In this form of leukemia we find a much greater anemia than in the 
splenomyelogenous form, although we may observe a normal red count for 
some time. The number of cells varies between 1,500,000 and 4,000,000, 
while the average percentage of hemoglobin is about 37 per cent. Nucleated 
red cells are rare in this condition, yet in the severer cases we may find them as 
numerous as in the splenomyelogenous type. All forms of degeneration noted 
under the previous type of leukemia are occasionally seen in this latter form. 

Leucocytes. 

The leucocytes are, as a rule, increased, the average being about 145,000, 
according to Osier. In this form we may find aleukemic periods which may 
last for a considerable period of time, the count usually rising just before death, 

Differential Count. 

According to Grawitz the cases of lymphatic leukemia may be divided 
into (1) those in which the increase of leucocytes is especially in the small 
mononuclear variety, (2) those showing an increase in the medium-sized 
cells with basophilic homogeneous protoplasm, and (3) those in which the 
cells which predominate are very large and usually degenerated. All these 
forms may occur together and may vary in the same case at different times. 
These mononuclear cells may constitute as high as 99 per cent. (Osier) of the 
total number of leucocytes. These leucocytes show in a very large number 
of cases much degeneration either of the protoplasm or of the nucleus, very 
few of the cells showing mitosis which is so common in the splenomyelogenous 
form. In this type of leukemia polymorphonuclear cells are rare, eosinophiles 
usually absent, and myelocytes and basophiles rarely if ever present. This 
type of leukemia is not easily amenable to diagnosis, especially in differentiating 
it from some cases of sarcoma in which the blood may show a similar picture. 

In some cases we find a lymphatic leukemia with a considerable number 
of myelocytes both of the eosinophile and neutrophile type. This has led 
to the differentiation of a " mixed leukemia," which does not seem to be advis- 
able as we may find myelocytes in the pure lymphatic type of this disease. 

(3). Acute Leukemia. 

This form of leukemia is characterized by its brief course (from six to 

eight weeks), by the severity of its symptoms, the frequency of the hemorrhagic 

1 See Nakamura, Deutsch. Ztschr. f Chir., 1914, CXXXTI, 275; Jackson and Smith, Boston 
Med. and Surg. Jour., 191 5, CLXXII, 136. 



544 DIAGNOSTIC METHODS. 

diathesis, rapidly developing cachexia, and death. This condition occurs 
chiefly in young people and is usually of the lymphatic type, 1 although a few 
cases of the myelogenous variety have been reported (Billings and Capps). 2 
In all cases the anemia is extreme, the red cells usually running below 1,000,000 
and the hemoglobin as low as 10 per cent. There is no type of cell which 
is characteristic of this form, although the cells are much more uniform in size 
than in the chronic states of this disease. In some cases nearly all of the cells 
have a basophile protoplasm, while in others they show acidophilic properties. 
Nucleated reds are usually rare, although they may be present in fairly large 
numbers. The drop in the count of red cells is usually sudden and denotes 
rapid blood destruction. The leucocyte picture resembles closely that of 
acute infections. 

(C). Pseudoleukemia. 
Under the heading Pseudoleukemia have been grouped a great variety of 
diseases, which have in some cases the external appearances of the disease, 
such as the glandular swelling, splenic tumor and progressive cachexia without, 
however, showing the blood picture which is so characteristic of leukemia. 
On the other hand, we find conditions which have little in common with the 
clinical findings of leukemia and yet show a blood picture similar in some 
respects to that of leukemia. It is difficult to group all of these cases under one 
heading, as the blood-picture is not characteristic for any one of these conditions. 

(1). Hodgkin's Disease. 

This condition, first described by Hodgkin in 1823, is characterized by 
chronically progressing cachexia with enlargement of the lymph-glands and 
spleen. It has been called, synonymously, lymphatic pseudoleukemia, 
lymphosarcoma, malignant lymphoma, lymphatic anemia and aleukemic myelo- 
sis. 3 It is probably infectious in nature, the etiologic factor being probably 
the Bacillus hodgkini (see Chap. XI). The blood characteristics in this 
disease are more particularly those of a true cachexia, the red cells showing 
a diminution in the number which may be as low as 2,200,000, but which 
is usually between 3,000,000 and 4,000,000. The more severe and pro- 
nounced the signs of anemia and cachexia the lower the number of cells, the 
number in these cases running as low as 1,500,000. Morphologically, the 
red cells show much less deviation from the normal than in other severe 
anemias, the average size of the cell being usually normal, degenerations of 
the red cells appearing only in the very severe conditions, microcytes aDd 
macrocytes, as well as nucleated erythrocytes, being very unusual except in 
the late stages. The hemoglobin content runs parallel to the number of red 
cells, being the lowest in those cases showing very low counts. The leucocytes 
are slightly increased, averaging about 12,000. This failure of a leucocythemia 

1 See Strauch (Am. Jour. Dis. Child., 1913, V, 43) for a discussion of this condition in 
children. Also, Beltz, Deutsch. Arch. f. klin. Med., 1913, CXIII, 116; Ballagi, New York 
Med. Jour., 1914, XCIX, 68; Yamakawa, Mitt. a. d. Med. Fak. Tokyo, 1914, XI, 115; 
Citron, Berl. klin. Wchnschr., 1914, LI, 332; Deutsch. med. Wchnschr., 1914, XL, 629. 

2 See Beifeld, Arch. Diag., 191 5, VIII, 244. 

3 See Hirschfeld, Ztschr. f. klin. Med., 1914, LXXX, 126; Ghedini, Gazz. d. osp., 1914, 
XXXV, 2037; Ibid., 1915, XXXVI, 1, 33, and 593; Ragusin, Sem. Med., 1915, XXI, 57 



THE BLOOD. 545 

enables us to differentiate Hodgkin's disease from a true lymphemia. The 
differential count of the leucocytes may show a relative lymphocytosis, the 
relation of the lymphocytes to the polynuclear cells being as three to one 
instead of the normal one to three. 1 Ehrlich and Pinkus consider this relative 
increase of lymphocytes characteristic of true pseudoleukemia in contradis- 
tinction to sarcomatous and other lymphomatous conditions. According 
to Grawitz, an increase in the leucocytes is associated with an unsatisfactory 
course of the disease, while a diminution in the number is observed as the 
disease progresses toward convalescence. 

Cases have been reported which would seem to point to the transition 
of Hodgkin's disease into a true leukemia, so that we may find irregular prog- 
ress of a pseudoleukemia as an evidence of a transitional stage. 

(2). Tuberculosis of the Lymph -glands. 

Why this condition has been classed as a pseudoleukemia is questionable, 
as the blood picture shows nothing beyond a secondary anemia with cachexia 
or may show even a normal red and white count. A large increase in 
the number of leucocytes, which is so characteristic of leukemia, is rarely 
seen, although a true leukemia may arise in the course of a glandular 
tuberculosis. The differentiation of this condition should be based upon 
examination of an excised gland, which will show distinct tuberculous 
lesions and usually will contain the tubercle bacilli in demonstrable num- 
bers. 2 In other cases we may find a simple lymphoid hyperplasia without 
any distinct inflammatory changes and without demonstrable bacilli. Such 
nodes should be tested by inoculation experiments as advised by Ewing. 
Heredity plays a great role in the diagnosis of these tubercular conditions, 
while a scrofulous child should always be looked upon with suspicion. A 
splenic tumor appearing coincidently with the glandular swelling would speak 
rather against tuberculosis and in favor of a lymphatic pseudoleukemia. 
The diagnosis of this condition by examination of the blood alone is a practical 
impossibility. It is this type of case that is especially amenable to diagnosis 
by the use of the various tuberculin tests. 

(3). Lymphosarcoma. 

The lymphosarcomata usually run their course either as primary benign 
lymphomata or as the malignant sarcomata. The blood findings in these 
conditions show nothing beyond a slight anemia with nothing characteristic 
in the appearance of the white cells. The diagnosis must be based entirely 
on the examination of the excised gland or tumor. 3 

(4). Gummatous Lymphoma. 

An exact diagnosis of this syphilitic swelling of the lymphatic glands is 
at the present time a matter of more or less difficulty. The previous history, 

1 Bunting (Bull. Johns Hopkins Hosp., 19 11, XXII, 114 and 369) considers an increase 
in the number of transitional cells together with increase of blood platelets as quite character- 
istic of true Hodgkin's disease. See, also, Bunting, Bull. Johns Hopkins Hosp., 1914, XXV, 
173 and 177; Yates, Ibid., 180. 

2 Resort should be made to the antif ormin method, if ordinary staining processes are 
not conclusive. 

3 See Oliver, Jour. Med. Research, 1913, XXIX, 191; Steiger, Berl. klin. Wchnschr., 
1913, L, 2129. 

35 



546 DIAGNOSTIC METHODS. 

as well as other manifestations of syphilis,, must be studied and a careful 
search made for the presence of the spirochgeta pallida. It is to be said that 
these organisms have been frequently reported in lymphatic enlargements which 
seem to have no direct relationship to purely syphilitic conditions. The 
interpretation of one's findings is of the utmost import as artefacts, which 
commonly appear in preparations of broken-down glandular tissue, resemble 
very closely the spirochete. The blood condition shows nothing characteristic 
and can have only incidental diagnostic importance. Application of the 
Wassermann serum test might throw much light on the diagnosis. 

(II). General Pathology. 
(a). Blood Changes Following Surgical Intervention. 

Under this heading the writer will not attempt to take up all of the surgical 
conditions, as the vast majority are not associated with any direct hematological 
characteristics. 

As a rule, it may be said that pus formation anywhere in the system will 
cause a leucocytosis. The degree of this leucocytosis averages about twice 
the normal standard, but may greatly exceed this figure in individual cases. 
It must be said, however, that trivial as well as extensive pus formations may 
be accompanied by normal or even subnormal values for the leucocytes. 
This is due to the facts that small pus foci do not cause any systemic reaction 
and extensive pus formation may overcome the power of the system to react 
against the infection. If the pus cavity is well encapsulated, the absorption 
of the toxin from this focus is necessarily limited so that we may find no leucocy- 
tosis, even though a very large pus cavity is present. Thus we find in localized 
peritonitis following appendicitis that a leucocyte count may be normal, 
but may suddenly increase to a marked extent as an indication of the rupture 
of the cavity and an extension of the process. 1 A general rule is that a distinct 
increase in the number of cells in excess of the figure originally obtained is 
indicative of the extension of pus formation and should put the surgeon on his 
guard as to operative interference. 

If the absorption of toxic material from a focus of pus formation is great 
enough to produce a systemic effect upon a patient as manifested by a high 
leucocyte count, we find an anemia characterized by a marked diminution in 
the hemoglobin and the number of red cells, which is parallel in intensity to 
the severity of the poisoning. 

Cases are frequently found in which a low leucocyte count prevails, although 
clinical evidence of severe sepsis is at hand. In these cases a differential 
count of the leucocytes should be made in all cases, as the low leucocyte count 
may throw one off his guard unless this precaution be taken. Should the 
polynuclear neutrophiles form 80 to 95 per cent, of the total leucocytes, 
a severe infection is indicated, even though the leucocyte count may be 
subnormal. 

If a leucocyte count does not diminish or even return to normal within one 
to two days after operation, this should be taken as evidence that a reinfection 

1 See Friedman, Am. Jour. Med. Sc, 1914, CXLVIII, 540. 



THE BLOOD. 547 

has occurred or that the pus cavity has not been properly drained. Recourse 
must, therefore, be had to measures to overcome the secondary infection. 

It has been found that administration of ether and chloroform causes 
a leucocytosis which usually lasts from 24 hours to 48 hours and which may 
interfere with the interpretation of a blood examination. This point must be 
borne in mind in the examination of blood of cases which have shown high 
leucocyte values prior to operative procedure. The differential count in such 
cases, however, will show only slight variations in the proportions of the dif- 
ferent types of cells so that one may judge as to the cause of the secondary 
leucocytosis by determination of the polynuclear cells. The number of red 
cells as also the amount of hemoglobin are very markedly reduced in some 
cases following the administration of an anesthetic, so that a direct secondary 
anemia may be the result. This fact has led to the refusal by many surgeons 
to resort to operative procedure in cases which show as low as 30 per cent, 
hemoglobin prior to operation. 1 

As the blood changes in surgical conditions, which are not accompanied 
by pus formation, are not especially characteristic and are more especially 
associated with the diseases of the special organs, the writer will refer such 
discussions to other headings. 

(&). Blood in Constitutional Diseases. 

(1). Diabetes Mellitus. 

In diabetes mellitus the changes in the cellular content of the blood are 
not very marked. The leucocytes may be subnormal, normal, or slightly in- 
creased, usually a very slight leucocytosis being observed. The amount of 
hemoglobin is usually reduced to a very slight extent, while the number of red 
cells may be slightly increased. 

One of the most striking peculiarities of the blood in diabetes is the presence 
of an excess of fat (lipemia). Microscopic examination usually reveals the 
presence of the extracellular globules, but in some cases fat is present in 
sufficient amount to permit of demonstration by macroscopic methods. Glyco- 
gen has been found both in the plasma and in the leucocytes of diabetic blood 
and shows the peculiar characteristics discussed in the section on Iodophilia 
(p. 509). Certain peculiarities of the blood in diabetes have led to the in- 
troduction of tests supposed to be characteristic for such blood. These tests 
are occasionally of diagnostic value, although a diagnosis may, as a rule, be 
made even when these tests do or do not obtain. 

Bremer's Test. — This test is based upon the fact that diabetic blood 
does not stain to any appreciable extent when treated with certain aniline 
dyes. Thick smears of the blood are made upon slides and are fixed by dry 
heat. These smears are then covered with a 1 per cent, aqueous solution 
of Congo red and allowed to stain for a few minutes, after which they are 
rinsed in water and dried. Diabetic blood will be stained either a faint yellow 
or not at all, while normal blood will be colored a bright red. A 1 per cent, 
solution of Biebrich scarlet will stain the diabetic blood intensely while the 

1 See Cullen, Surg., Gynec. and Obst, 1913, XVII, 276. 



548 DIAGNOSTIC METHODS. 

normal blood is unstained. Bremer's original staining solution was made 
up as follows: Saturated watery solution of eosin and of methylene blue are 
mixed in equal proportion when a precipitate forms which is filtered, washed, 
dried and powdered. To 24 parts of this powder are added six of powdered 
methylene blue and one of eosin. One-twentieth of a gm. of this mixed 
powder is dissolved in 10 c.c. of 33 per cent, alcohol and forms the staining 
solution in which the specimens are stained for four minutes. The diabetic 
blood stained by this solution has a greenish tint, while normal blood is 
reddish violet. 

Similar reactions have been found in normal blood, in leukemia, in exoph- 
thalmic goiter, in Hodgkin's disease, in multiple neuritis, and in some cachectic 
conditions, but the reaction in all these cases is very inconstant. According 
to Bremer, cases of renal diabetes do not give this reaction, which is more 
characteristic of the pancreatic type of the disease. 

Williamson's Test. — This test is performed as follows: Two drops of 
blood (20 cmm.) are dissolved in four drops (40 cmm.) of water and to the 
solution is added 1 c.c. of a 1 to 6,000 aqueous methylene blue solution. 
To this is added four drops (40 cmm.) of 6 per cent, solution of liquor potassae 
and the test-tube placed in boiling water for four minutes. Diabetic blood 
will decolorize the solution, while normal blood leaves it a deep blue. The 
same effect is observed by using diabetic urine instead of the blood. 

(2). Gout. 

Little information is available as to the variations in the number of the 
red and white cells in the gouty condition. The recorded observations show 
that acute gout has little effect upon the number of red cells and upon the 
amount of hemoglobin, while chronic gout may be accompanied by an anemia 
which is more directly referable to causes other than the gouty condition itself. 
The leucocytes are usually increased in the acute attack, while in the chronic 
form the leucocytosis is of a more moderate grade. Neusser in working upon 
the blood of gouty patients found many polymorphonuclear leucocytes whose 
nuclei were surrounded by basophile granules — the so-called perinuclear baso- 
phile granules. These he considered diagnostic of the uric acid diathesis, but 
Futcher and Simon have found them in many other conditions, while Ehrlich 
regards them as artefacts. 

While the chemistry of the blood in gout has been the subject of much 
investigation for a long period, nothing of diagnostic importance has been found 
in the chemical properties of the blood. The excess of uric acid has been 
shown not to be pathognomonic of gout, as it is present in many other conditions 
which are clinically far removed from the gouty state. 

(3). Addison's Disease. 

This disease is usually associated with a severe grade of anemia, the 
number of red cells being reported as low as 1,120,000 while the percentage 
of hemoglobin is coincidently reduced. The leucocytes are usually diminished, 
but may be slightly increased, while the relative proportions of the different 



THE BLOOD. 549 

cells are not markedly changed. As the disease progresses unfavorably, a 
relative lymphocytosis may be observed, but this is not always the case. 1 

(4). Rickets. 

The state of the blood in rickets varies with the extent and severity of the 
primary disease and is markedly affected by complications. Cases are re- 
ported in which the red cells are practically normal and in which the hemoglobin 
was only very slightly reduced. This disease is not associated with any special 
type of anemia, although the hyperemia of the bone-marrow might be expected 
to yield a large number of nucleated red cells. The usual condition of the 
blood in rachitic children is of the type of simple chlorotic anemia. A grave 
secondary anemia is seen in many cases in which there are serious complica- 
tions. The leucocytes in practically all cases of rickets are increased, but 
may not exceed the normal limits for the child. As is usual in the blood of 
a child, the lymphocytes are increased while the eosinophile cells are often 
relatively numerous. 2 Just exactly what the cause of the leucocytosis in 
rickets is must be left to the realm of hypothesis, as neither the gastroenteritis 
nor the hyperplastic splenitis are sufficient to explain all cases. 

(5). Myxedema. 

In this disease we usually find an anemia of the secondary chlorotic type 
along with a moderate leucocytosis. The number of cells is usually somewhat 
diminished, although their size is usually increased. The proportion of the 
different leucocytes does not vary, although a few myelocytes are sometimes 
seen in the blood, which is numerically normal in other respects. 

Although many studies of the chemistry of the blood in myxedema have 
been made, little knowledge has been forthcoming as to the exact cause of 
this toxemia. It is highly probable that the changed activity of the thyroid 
gland in this disease influences other organs to such an extent that slight 
anemia is the result and we should, therefore, assume that this anemia is more 
a secondary one than a primary result of thyroid insufficiency. 3 

(c). Blood in Acute Infectious Diseases. 

In a study of the blood in acute infectious diseases, we must remember 
that there are certain general rules which apply to all of such diseases with 
a very few exceptions. The interrelation of the fever with the resistance of 
the system in general is so close that it is hard to say in any given case whether 
certain changes in the blood are or are not due to the increased temperature 
in itself. There can be little doubt that a high temperature working over a 
considerable period of time will destroy large numbers of red cells and will 
bring on various changes in the blood which might be misinterpreted. A 
rather extensive concentration of the blood along with a progressive loss of 
albumin is observed in practically all conditions associated with fever. We 

1 See Buettenmiiller, Med. Klin., 1914, X, 1354; Engelsmann, Folia Haemat., 1915, XIX, 
333; Scheltema, Xederl. Tijdschr. v. Geneesk., 1915, I, 1767. 

2 See Ostrowski, Folia Haemat, 1912, XIII, 305. 

3 See Salvatore (Riforma Med., 1913, XXIX, 1373) for a discussion of the blood changes 
in exophthalmic goiter. 



550 DIAGNOSTIC METHODS. 

should, therefore, expect to find the number of red cells increased at the outset 
of such condition, owing to the concentration of the fluid portion, while a dis- 
tinct anemic condition may become evident only after the lapse of some time. 

Fever in itself does not have a large influence upon the number of leuco- 
cytes, but it may be stated as a general rule that most infectious diseases (the 
exceptions being malaria, typhoid, tuberculosis, influenza, and measles) are 
associated with an increase in the number of white cells. This is not an 
invariable rule, as will be seen under the discussion of the various infectious 
types. The leucocytosis so commonly seen associated with infection is no 
doubt due to the action of the bacteria themselves and of their products 
upon the leucocytes. The positive chemotaxis which bacteria and their 
toxins exert upon the leucocytes is very marked. Moreover, as the blood 
becomes laden with these abnormal products, new leucocytes are thrown 
into the circulation to aid the old ones in their phagocytic action. Just what 
substances are accountable for the increased opsonic power of the serum in any 
specific infection must be left undecided for the present. 

In the following discussion of the various infectious diseases, the writer 
will not attempt to give more than a brief discussion of the blood changes 
in these separate conditions, leaving associated questions to other writers. 

(i). Pneumonia. 

This disease is, hematologically, one of the most definitely characterized 
of all the infectious diseases. While the physical findings of this condition 
are largely local, the systemic effects are so marked that definite changes are 
seen in the blood both in the early and in the later stages of the infection. 
While showing so many characteristic findings in the blood, it is, at the same 
time, one of the most obscure in its relations to opsonins and to phagocytosis. 
Just why the virulent pneumococci should be so little capable of phagocytosis 
and why the addition of attenuated cultures of pneumococci or of extracts of 
virulent organisms should increase this phagocytic power of the leucocytes 
is at present very uncertain. We must, therefore, leave the discussion of this 
phase of pneumonia as well as of other infectious diseases to the section on 
Bacteriology of the Blood. 

In pneumonia we find in the early stages that the blood is somewhat 
concentrated owing to the action of the increased temperature, while this 
concentration gradually increases as the exudate forms. Such a condition 
can lead only to an increased count of both the red and the white cells. As 
the disease progresses, the number of red cells shows a slight but a steady 
decline, which points not only to a destruction of the red cells, but to a diminished 
formation. This decrease in the number of red cells is occasionally seen only 
at the time of crisis, while in the cases in which the diminution is gradual the 
period of diminution does not usually exceed ten days. It will be seen, there- 
fore, that the red cells in pneumonia may be about normal in number and at 
the same time an anemia may be present which becomes evident only after 
the disease has progressed for some time. The red cells are, as a rule, normal 
in appearance, but an occasional polychromatophilic cell may be seen, especially 






THE BLOOD. 551 

in the severe cases. Rarely normoblasts may be observed and very rarely 
megaloblasts. > 

The hemoglobin usually shows a greater reduction than does. the number 
of red cells, which decrease may become evident only after the fever has sub- 
sided. A reduction in the hemoglobin below 60 per cent, is very unusual in 
pneumonia of the pure type. 

Pneumonia is one condition in which the leucocyte count may prove of 
great value. A leucocytosis appears in most of the cases, being absent in 
very mild cases as well as in those very severe ones which show very feeble 
resistance of the organism toward invasion by the pneumococci. 1 Rieder's 
observations are very interesting on this subject. He has found that the 
leucocytosis of pneumonia is more a function of the intensity of the infection and 
the degree of resistance toward this infection than it is of the fever or of the 
extent of the exudate. 2 A leucocytosis which may reach 12,000 to 20,000 
appears very early in the course of this disease, and is usually evident at the time 
of the chill or immediately following. A steady increase is sometimes observed 
in the number of the white cells so that the maximum is usually reached just 
before the crisis. It is to be said that rapid extension of the disease as well as 
continuous high temperature may cause much irregularity in the count, cases 
being reported in which the leucocytes are high at first and steadily diminish 
as the patients grow worse. Others may show a sudden increase in the number 
of cells as the time of crisis is approached. 3 According to Ewing,when the leu- 
cocytes increase slowly they usually diminish slowly and the disease defervesces 
by lysis. The degree of leucocytosis in pneumonia may reach any stage be- 
tween the normal figure and that of 115,000 as reported by Laehr. 

The increase in the number of white cells in pneumonia is largely referable 
to increase in the number of the polynuclear neutrophiles, these cells con- 
stituting as high as 97 per cent, of the total number of white cells. Associated 
with this polynuclear leucocytosis we have a marked diminution of lympho- 
cytes, while the large mononuclear cells usually persist in considerable numbers. 
The eosinophile cells are always much reduced at the height of the leucocytosis, 
so that we may not be able to find a single one after very prolonged search. 
Cabot has reported a case in which the lymphocytes constitued 66 per cent, 
of a total of 94,600 white cells, but such a finding is not the usual one fol- 
lowing infection with the pneumococcus. As defervescence goes on the 
polynuclear cells diminish very rapidly, while the lymphocytes increase and 
the large mononuclear leucocytes become very numerous, reaching as high as 
16 per cent, in a case reported by Turk. The eosinophile cells usually appear 
about the time of crisis, but occasionally their appearance is postcritical. The 
degenerative changes seen in the leucocytes in pneumonia are in no way 
different from those observed in other infectious diseases. 

It will be noted, from the above remarks, that the blood changes in pneu- 
monia are those of a mild anemia associated with a high-grade polynuclear 

1 See Hess, Am. Jour. Dis. Child., 1914, VII, 1. 

2 See Kline and Winternitz, Jour. Exper. Med., 1913, XVIII, 50. 

3 Dick (Jour. Infect. Dis., 1912, X, 383) shows that proteolytic ferments develop in the 
blood about the time of crisis. 



552 



DIAGNOSTIC METHODS. 



leucocytosis and a distinct lymphopenia. Too much reliance must not be 
placed on the blood finding in a case of pneumonia, owing to the fact that 
many abnormal cases are present and show results different from the above 
which can be interpreted only by a complete study of the complications in 
any special case. As a general rule, it is to be said that an absence of leucocy- 
tosis is strong negative evidence against pneumonia, while leucocytosis may 
serve to differentiate this condition from typhoid fever and malaria with which 
it might be confounded, especially where the systemic and cerebral symptoms 
are more pronounced than are the local pulmonary changes. 

(2). Typhoid Fever. 

This condition, like the preceding, is very often associated with such marked 
systemic disturbance that the local intestinal manifestations are obscured 
and the diagnosis rendered somewhat difficult. While typhoid fever is a 
purely infectious condition and subject to the ordinary laws governing such 
cases, yet we find for some reason that the invasion of the blood by the specific 
organism is not associated with a leucocytosis, although the febrile rise may 
be very marked. The study of the characteristic serum reaction for typhoid 
fever as well as of the bacteriology of the blood must be left for a later section, 
the discussion here being limited to the changes in the microscopic appearances 
of the blood. 

In this condition we find the total volume of blood very much diminished 
in the early stages, both as a result of the high temperature and the diarrhea 
and repeated hemorrhages which may occur at any stage of the disease. This 
concentration of the blood leads to an initial polycythemia which may last for 
two or even three weeks However the characteristic change in the red cells is 
one of a slight and gradual decline, the number of these cells not usually falling 
below 4,000,000. One must be on his guard in an examination of the blood 
in any infectious disease lest he conclude from a slightly increased count that 
no anemia is present. It is a very safe precaution, although rarely followed, 
to determine the specific gravity of the blood so that one may compute the 
degree of concentration. In this way he may be able to show that the number 
of cells normally present in such a concentrated blood is much higher than in 
the case of suspected typhoid fever which he is examining. At any rate, it is 
wise to make frequent determinations of both the number of cells and of the 
hemoglobin, as a reduction in both of these elements takes place gradually 
as in the cases reported by Thayer and Da Costa. 

The reduction in the amount of hemoglobin is in some cases very marked, 
being as low as 50 per cent, in one case observed by the writer. The repeated 
hemorrhages which so often occur in typhoid fever may cause marked varia- 
tion in this value. The morphological changes which occur in the red cells 
are not very marked, as a rule, but may be very severe in case much blood is 
lost by frequent hemorrhages. Polychromatophilia is more or less frequent 
and irregularity in the size of the red cells is occasionally seen, while nucleation 
of these cells along with formation of a few megaloblasts may occur in severe 
hemorrhagic cases. 



THE BLOOD. 553 

The leucocytes are usually normal in number in the early stages of uncom- 
plicated cases, but any complication may cause a polynuclear leucocytosis 
which may be confusing to the worker. The behavior of the leucocytes is 
very variable in the early stages, so that one should never rest his diagnosis 
of typhoid fever upon a negative leucocytosis. As the disease progresses, the 
leucocytes show a gradual reduction, especially in the number of the polynu- 
clear cells, which reduction continues until the disease has reached its highest 
point, after which they slowly increase. 

The more severe the action of the typhoid toxin the lower is the leucocyte 
count, the reduction not usually going below 2,500 cells, the majority of cases 
showing a count between 4,000 and 6,000. It is not an uncommon thing to 
observe a leucocytosis during the later course of typhoid fever and it is not 
always easy to explain such a condition. Marked hemorrhage, cold baths, 
severe diarrhea, and usually perforation may account for the increase in the 
number of cells, but we do not always find a leucocytosis after such conditions. 

In typhoid fever we find quite characteristic changes in the relations of 
the various types of leucocytes. During the first week the neutrophile cells 
do not, as a rule, increase, while the lymphocytes, especially of the medium-size 
variety, show a progressive rise. The lymphocytes are rarely below 25 per 
cent, of the total number of cells and may reach as high as 65 to 70 per cent. 
The eosinophile cells are usually low in number during the febrile period, but 
reappear about the time of defervescense. 

It will thus be seen that the characteristic changes in the blood of typhoid 
fever are a slight anemia, together with a leucopenia and a relative, and in 
some cases absolute, lymphocytosis. Such characteristics are the usual ones 
of typhoid fever, but it is to be remembered that suppurative processes do not 
always produce a leucocytosis, nor is a leucopenia always present in typhoid 
fever. 

(3). Scarlet Fever. 1 

In this condition we find the usual effects of fever manifested in a slight con- 
centration of the blood, leading in the early stages to a polycythemia. The 
usual change, however, in the red cells is one of gradual reduction in numbers 
to as low as 3,000,000 cells and occasionally much lower. The hemoglobin 
also suffers quite a diminution, so that the anemia may reach quite a severe 
grade. 

The leucocytes in scarlet fever usually increase in number one or two 
days before the appearance of the rash and continue to increase until quite 
a marked leucocytosis, ranging from 10,000 to 50,000 cells, becomes evident 
at the time of the complete eruption. The degree of the leucocytosis cor- 
responds as a rule with the severity of the disease and in some cases is diminished 
at the time of the eruption, but usually continues for several days and may 
even extend for weeks after the temperature has subsided. 

The increase in the number of leucocytes is largely referable to the poly- 
nuclear cells, these constituting from 85 to 99 per cent, of the total number. 

1 See Koessler (Jour. Am. Med. Assn., 1912, LLX, 1528) for a discussion of the recent 
advances regarding scarlet fever. 



554 DIAGNOSTIC METHODS. 

The lymphocytes diminish in the early stages of the disease, but later rise to 
normal or slightly above normal figures. The eosinophile cells are usually 
normal or even subnormal at first, but steadily increase as the disease pro- 
gresses and reach a degree of 10 to 20 per cent, in the second or third week, 
after which they slowly decline. These rules are not invariable in scarlet 
fever, but a severe leucocytosis appearing prior to the period of eruption of an in- 
fectious fever is practically always suggestive of this disease. In some cases the 
polynuclear cells diminish about the end of the first week and the lymphocytes 
and eosinophiles rapidly increase, leading to a later secondary leucocytosis. 1 

(4). Measles. 

This condition shows in itself nothing particularly characteristic in the 
blood, but the absence of definite findings is of great importance in its differen- 
tiation from scarlet fever with which it might be confounded. The red cells 
in this disease are not found to be greatly changed, although a slight reduce 
tion in their number is usual. A loss of hemoglobin is practically always 
noticed. 

The leucocytes are usually normal or slightly reduced in number at the 
outset of the disease, being the lowest at the height of the eruption when the 
figure may reach as low as 2,500 cells, returning to the normal within a few 
days after subsidence of fever. A complicating bronchitis may cause a moder- 
ate leucocytosis of 8,000 to 16,000 cells, but this should not lead one to a mis- 
taken diagnosis, as the clinical symptoms of both scarlet fever and of measles 
should be well-established at the time of the complicating bronchitis. An 
eruptive fever in the second or third day of its course should be considered 
scarlet fever, or at least scarlatina, if a leucocytosis is present, while if the 
disease be measles the number of leucocytes will be normal or even subnormal 
in the absence of extensive bronchitis. 2 

1 Dohle (Centralbl. f. Bakteriol., 191 1, LXI, 63; Ibid., 1912, LXV, 57; Munch, med. Wchn- 
schr., 1912, LIX, 1688) has announced the almost constant presence in the polymorphonuclear 
leucocytes of " inclusion bodies." These are observed near the margin of the cell as rod or 
coccus forms, which stain readily a faint bluish-green tint with the Giemsa or other blood stains. 
While these bodies are observed in about 95 per cent, of scarlet fever cases in the early days 
of the disease, the number diminishes gradually. They are also found in many cases of diph- 
theria, pneumonia and tuberculosis, yet they are very infrequent in cases of serum sickness or 
of scarlatiniform rashes. Their presence is, therefore, not pathognomonic but their absence 
almost excludes scarlet fever. See Kretschmer, Berl. klin. Wchnschr., 1912, XLLX, 499; 
Deutsch. med. Wchnschr., 1912, XXXVIII, 2163; Nicoll and Williams, Arch. Pediat., 1912, 
XXIV, 350; Ahmed, Berl. klin. Wchnschr., 1912, XLIX, 1232; Kolmer, Am. Jour. Dis. Child., 
1912, IV, 1; Granger and Pole, Brit. Jour. Child. Dis., 19135X5 9; Lippmann and Hufschmidt., 
Zentralbl. f. inn. Med., 1913, XXXIV, 369; Schippers and de Lange, Berl. klin. Wchn- 
schr., 1913, L, 544; Bongartz, Ibid., 544; Schwenke, Munch, med. Wchnschr., 1913, LX, 752; 
Pappenheim, Folia Haemat., 1913, XV, 379; Cummins, Jour. Med. Research, 1913, XXVII, 
529; Nicoll, Arch. Pediat., 1913, XXX, 346; and Brinchmann, Berl. klin. Wchnschr., 1913, 
L, 1248; Massini, Med. Klin., 1913, IX, 1729; MacEwen, Jour. Path, and Bacteriol., 1914, 
XVIII, 456; Rosanoff Arch. f. Kinderhkde., 1914, LXII, 321; Hill, Boston Med. and Surg. 
Jour., 1914, CLXX, 792; Isenschmid and Schemensky, Munch, med. Wchnschr., 19145 LXI, 
1997; Kaiser and Lowy, Deutsch. Arch. f. klin. Med., 1914, CXVI, 82; Rehder, Ibid., 1914, 
CXVII, 37. 

2 Schwaer (Munch, med. Wchnschr., 1913, LX, 1203) believes that a diminution or dis- 
appearance of eosinophiles at the height of the fever is characteristic of measles. See, also, 
Lucas, Am. Jour. Dis. Child., 1914, VII, 149; and Grumann, Munch, med. Wchnschr., 
1914, LXI, 132. Hess (Arch. Int. Med., 1914, XIII, 913) discusses the blood picture in 
German measles. 



THE BLOOD. 555 

(5). Variola. 

This condition is associated with more or less extensive destruction of 
the red cells. 1 In the early stages of the disease the red count may be slightly 
above normal, owing to the concentrating effect of the fever, but later the red 
cells will show a sudden reduction. This reduction is especially noticeable 
in the cases associated with extensive pustulation, when the septic process has 
such a marked influence in destroying the red cells. The hemoglobin is 
usually reduced in degree parallel to the diminution in red cells. 

In most of the cases of small-pox we find a distinct leucocytosis which 
may run from 10,000 to 20,000 as a rule, but has reached as high as 41,000 
in the severe cases. This leucocytosis begins with the appearance of the vesicle, 
increases as the exudate becomes purulent, and reaches its height when sup- 
puration becomes extensive; that is, the degree of leucocytosis usually runs 
parallel to the severity of the septic process, the count returning gradually 
to normal as the suppuration subsides. The leucocytosis in small-pox is 
usually of the lymphocytic type, the number of these cells varying from 35 to 
45 per cent, of the white cells. Associated with the increased lymphocyte 
value we find from 5 to 10 per cent, of the large mononuclear leucocytes and 
usually an average of 3 per cent, of neutrophile myelocytes, in some cases 
these latter running as high as 16 per cent. Eosinophiles and basophiles 
are occasionally observed, especially in the hemorrhagic form of this 
disease. 

It will thus be seen that small-pox causes a leucocytosis which may reach 
even the degrees given by scarlet fever, but the differential count as shown by 
the large percentage of lymphocytes in the former and the greatly increased 
number of polynuclear neutrophiles in the latter should make a mistake in 
diagnosis impossible. It is to be remembered that complications of true 
abscesses with the pustules of small-pox may increase the percentage of 
polynuclear cells in this disease, but never to such a degree as is shown in 
scarlet fever. 

An examination of the blood of children, who have been vaccinated with 
small-pox virus, shows a distinct leucocytosis of the polynuclear type, reaching 
as high as 20,000 cells. This leucocytosis usually begins on the third or fourth 
day after inoculation and gradually subsides until the end of the period of 
vaccination. 

The blood in cases of varicella 2 seems to show the same characteristics 
as that of very mild cases of variola, or of vaccinia. The slight leucocytosis 
is usually of the polynuclear type, reaching a degree of 15,000. The large 
mononuclear cells which seem to play such an important role in the differential 
leucocyte picture of variola are for the most part absent in varicella, and 
myelocytes are practically never found in this condition. 

1 See Proescher (Interstate Med. Jour., 1915, XXII, 427) for a discussion of the cultivation 
of the variola- vaccine virus. 

2 See Mensi, Gazz. d. Osp., 1912, XXXIII, 1625; also, Erlenmeyer, Deutsch. med. Wchn- 
schr., 1913, XXXIX, 21; Schatzmann, Cor.-Bl. f. schweiz, Aerzte, 1913, XLIII, 1515; Ztschr. f. 
klin. Med., 1914, LXXX, 333; Erlenmeyer and Jalkowski, Deutsch. med. Wchnschr., 1914, XL, 
646. 



556 DIAGNOSTIC METHODS. 

(6). Diphtheria. 

The number of red cells in diphtheria seems to be slightly increased, 
owing to the marked concentration of the blood in this condition. In practi- 
cally all cases of diphtheria the number ranges from a high normal value to 
as high as 7,800,000 reported by Cuffer. As the disease progresses, especially 
after the temperature has fallen, the number of red cells is diminished and 
a coincident decrease in the percentage of hemoglobin is observed. This 
slight anemia is not evident in the early stages of the disease due, no doubt, 
to the abnormal concentration of the blood. 

In this condition, like most infections, we find a leucocytosis ranging 
between 25,000 and 50,000, the higher the leucocytosis the more grave the 
prognosis. In one case reported by Felsenthal the leucocytes numbered 
148,000, but this is very unusual, as the grade of leucocytosis is ordinarily 
proportional to the extent and depth of the membrane. The leucocytosis 
is usually of the polynuclear type, the lymphocytes being also slightly in- 
creased. In some cases a lymphocytosis of 60 per cent. (Ewing) has been 
observed, but this is not the usual finding. The eosinophile cells are reduced 
in number, but are relatively more numerous than in pneumonia. It is in 
diphtheria that we find quite marked degenerative changes in the leucocytes, 
"the leucocyte shadows" and increased acidophile tendency of the neutrophile 
granules being especially worthy of mention. 

(7). Pertussis. 

The exact work of Barach 1 and others has so modified our ideas of the 
changes in the blood in pertussis that I can do no better than to give his 
summary upon these points: 

"In the early stages of this disease there is a leucocytosis with increase 
of all the forms; then a small-cell lymphocytosis becomes conspicuous and 
continues to increase when the other forms have reached their limit. The 
large lymphocytes follow the course of the small ones, but they reach their 
greatest numbers after the small cells have reached theirs. During the stage 
of active lymphocytosis, bilobed, small lymphocytes are frequently seen as 
well as numerous degenerated large lymphocytes, especially the basket forms. 
Then comes the simultaneous falling of the leucocytosis and lymphocytosis, 
while the polynuclears begin to resume their normal proportion. A little 
later the mast cells are observed more frequently, and an occasional myelocyte 
may be seen. While the leucocytosis and lymphocytosis continue to fall by 
lysis, an eosinophilia is noted; this continues for a variable time, after which 
the blood formula resumes its normal proportions. During this entire cycle 
the transitionals seem unaffected. 

If we were to speak of the first and second half of the blood cycle in this 
disease, we would say that in the first half the lymphocytes are the prominent 
factors and in the second half the polynuclears and the eosinophiles. 

Clinically, leucocytosis is present at about the time the child first coughs; 
as the coughing goes on, the leucocytosis increases and the lymphocytosis 

1 Arch. Int. Med., 1908, I, 602. See, also. Schneider, Munch, med. Wchnschr.. 1014, 
LXI, 303. 



THE BLOOD. 557 

becomes very marked. Churchill believes that a lymphocytosis exists in the 
prespasmodic stage and is of extreme importance in early diagnosis. The 
height of the leucocytosis is reached in the spasmodic stage, sometimes early, 
and sometimes in the latter part, the sickest children showing the highest 
grade of leucocytosis. About the time that a marked improvement is noted 
in the child the leucocytosis has decreased, the polynuclears have increased 
and the eosinophilia is present." 

The degree of leucocytosis in this condition varies between 2 5,000 and 51,150. 

(8). Acute Rheumatism. 

In this disease we find that the red cells are quite markedly destroyed, 
causing, very frequently, a reduction of 2,500,000 cells. This reduction is 
not always evident in the early stages of the disease, as the blood becomes 
very much concentrated by the marked sweating which is such a prominent 
symptom of the disease. The hemoglobin suffers more than the red cells 
so that we may find the percentage of hemoglobin as low as 60 per cent, in this 
condition. This anemia is one of the characteristic signs of acute rheumatism 
and continues well on into convalescence, the hemoglobin not being as quickly 
restored as are the red cells. A largely increased formation of fibrin has been 
observed and may have some diagnostic importance. 

The leucocytes are increased in proportion to the severity and acuteness 
of the disease, the grade being usually moderate and the type being poly- 
morphonuclear. In the very mild cases we may find no leucocytosis and one 
reaching 20,000 or more is, according to Tiirk, always associated with com- 
plications. As the fever diminishes the leucocytes return to normal and are 
not as much affected by the subsequent attack as by the previous initial one. 
The eosinophile cells are absent only in the early stages, while they are present 
in moderate amounts later in the disease and show a distinct increase after 
defervescence (Loeffler) . 

It would be impossible in a work of this character to discuss in detail the 
blood changes in all diseases, whether infectious or non-infectious. 1 The writer, 
therefore, has selected under the acute infectious diseases those which show 
the more characteristic changes in the blood and those in which an examination 
of the blood is more frequently called upon to aid in diagnosis. Many phases 
of these diseases have been left to the chapters on bacteriology of the blood. 

(d). Blood in Chronic Infections. 

(1). Tuberculosis. 

The earlier studies of the blood of tubercular patients reveal the fact 
that the blood may show practically no changes which are comparable with 
the pallor of the skin and the degree of the emaciation of the subjects affected. 
An anemia is often seen of the very highest type, but usually one of moderate 
degree is present and even may not exist at all. The degree of anemia is 
independent of the localization of the disease, although pulmonary affections 
are more frequently associated with high-grade anemia than are other tubercu- 

1 See Barach (Arch. Int. Med., 1913, XII, 751) for blood changes in mumps. Hess and 
Fish (Am. Jour. Dis. Child., 1914, VIII, 385) discuss the blood in infantile scurvy. 



558 DIAGNOSTIC METHODS. 

lar conditions. It is to be remembered that pulmonary tuberculosis is so 
frequently associated with extensive hemorrhage that one may not wonder 
at the severe anemia present, yet we find cases in which the regeneration is 
very rapid after severe hemoptysis. As a rule, a mild anemia of the chlorotic 
type prevails; that is, the count is practically normal with the hemoglobin some- 
what reduced. Occasionally we find a slight lymphocytosis, especially of 
the smaller cells, and only when a secondary infection prevails does the leuco- 
cytosis take on the polynuclear type. 1 The lymphocytosis in tuberculosis is 
so common that we usually find even in the sputum an excess of the small 
mononuclear cells in the pure tubercular affection of the lungs. 

In tubercular infection of the meninges we practically always find a 
leucocytosis, but with this exception uncomplicated tuberculosis is not asso- 
ciated with an increase in the number of white cells. It is highly probable 
that the increased percentage of the mononuclear cells is more closely associated 
with the poor nutrition which the tubercular patient shows than it is with 
any specific effect of the bacillus tuberculosis. Occasionally we find the 
eosinophile cells somewhat increased, especially in pulmonary conditions 
with cavity formation, but it must be remembered that a slight eosinophilia 
will obtain if tuberculin therapy is being used in such cases. 

(2). Syphilis. 

According to Becquerel and Rodier, a moderate grade of anemia is to 
be found in the majority of cases of syphilis, becoming more pronounced 
as the disease progresses. This anemia of syphilis is of the chlorotic type, 
but may increase until the pernicious type becomes established. As a rule, 
the reduction in the number of red cells is moderate, being rarely below 
3,000,000 cells. The hemoglobin is usually relatively more decreased than 
are the cells, and the application of mercury in the treatment of this condition 
frequently lowers this percentage still further, establishing an anemia which 
is directly referable to the mercury. This reduction in the number of red cells 
and in hemoglobin becomes more marked as secondary symptoms appear, 
so that a diagnosis of an initial lesion becomes established by a later examination 
of the blood. 

The increase in the number of leucocytes is largely limited to the secondary 
and tertiary stages of this disease, as the leucocytes usually remain normal up 
to the time of the eruptive stage. The increase in the secondary stage, which 
may reach as high as 20,000, is largely in the number of the small and large 
lymphocytes, but the eosinophile cells may be increased to as high as 5 per 
cent, in some cases. In very severe cases a progressive polynuclear leucocytosis 
is observed. As the tertiary stage comes on the leucocytosis usually persists, 
but the lymphocytosis becomes less distinct and constant through the increase 
in the number of polynuclear cells. 2 

1 See Miller and Reid, Arch. Int. Med., 1912, IX, 609; Miller, Lupton and Brown, Am. 
Jour, Med. Sc, 1912, CXLIII, 683; Ringer, Ibid., 1912, CXLTV, 561; also, Rayevsky, New 
York Med. Jour., 1913, XCVII, 813; Wack, Deutsch. Arch. f. klin. Med., 1914, CXV, 596. 

2 See Hazen, Jour. Cutan. Dis., 1913, XXXI, 618 and 739. 



THE BLOOD. 559 

Justus' Test. 

Justus has found, in studying the blood of patients suffering with florid 
syphilis, that injection or inunction of preparations of mercury cause a reduction 
in the percentage of hemoglobin of from 10 to 20 per cent, for a period of a 
few hours or days. After a certain time, varying with the general condition 
of the patient and the severity of the symptoms, the hemoglobin increases 
again. This test can hardly be considered diagnostic of syphilis, as the mer- 
cury salts all cause an anemia which may be directly traceable to their hemo- 
lytic action upon the red cells. 

In the blood of patients suffering with congenital syphilis we always find a 
distinct anemia, associated with a slight leucocytosis, especially of the lymph- 
ocytic type. The red cells in this condition show many changes, such as poly- 
chromatophilia and nucleation, while the changes in the white cells often 
resemble the picture of a mild grade of leukemia. For a discussion of the causa- 
tive factor, the spirochaeta pallida, see the section on Parasitology (p. 579). 

(3). Leprosy. 

The blood in leprosy is quite different from that in either of the two pre- 
vious conditions. The usual rule is a very slight reduction in the number 
of red cells, although cases have been reported with a red count of 1,906,000 
and a blood picture of pernicious anemia. The hemoglobin does not seem 
to be reduced to any extent, the percentage usually being relatively higher 
than the number of red cells, so that a high color index almost invariably 
obtains. The leucocytes are rarely increased in number, being usually sub- 
normal, with a relative increase in the number of lymphocytes, their percentage 
reaching as high as 47 per cent., according to Winiarski. 

(4). Carcinoma. 

Although discussed under the heading of Chronic Infections, carcinoma 
has at present no etiological relation to such conditions. This is one of the 
most important causes of anemia, owing to the frequent hemorrhages and the 
mechanical effects of the growth as well as to the unknown toxin, which may 
produce severe constitutional symptoms, even though the growth may have 
become latent. The anemia of malignant disease usually runs parallel to 
the progressive cachexia. The grade of anemia may vary, depending upon 
the location of the tumor, from a very mild chlorotic anemia to one with a 
perfect picture of pernicious anemia. It is natural to suppose that the more 
malignant the disease the greater will the blood changes be, so that we should 
expect to find the rapidly growing cancers which form numerous metastases 
usually associated with the more extreme blood picture. That this is not 
always the case is shown by the following statement of Emerson: "Our 
cases with rapidly developing metastases, with large nodules, are those with 
a slight chlorotic anemia; those which simulate pernicious anemia are more 
often those with few objective signs of cancer, an insignificant-looking little 
nodule." It is possible that this paradox may be explained by the fact that 
the development of the cancer is so rapid that the toxin has not had sufficient 
time to cause the blood changes which the more slowly developing growth 



560 DIAGNOSTIC METHODS. 

may bring about. The changes in metabolism are much more marked in 
the slowly developing cancers than in the more rapidly growing ones, so that 
we might assume that the same rule applies to the blood changes as expressions 
of the general systemic disturbances. 

When the anemia of cancer develops it is usually more severe than in any 
other chronic disease. The chief changes are at first in the size, shape, weight, 
and degeneration of the red blood-cells; later, as the cachexia develops the red 
cells are often as low as 2,500,000 or even as low as in pernicious anemia, 
1,000,000. The hemoglobin is always reduced in amount, but is rarely as 
low as in chlorosis, the average, according to Cabot, being about 58 per cent. 
The hemoglobin value seems to be lower in cases of visceral cancer than in 
those of peripheral type. In a majority of the cases a moderate leucocytosis 
obtains which is never seen in the benign tumors unless these be complicated 
by suppuration. This leucocytosis depends largely upon the amount of 
hemorrhage from the tumor and upon the position of the cancer. We find 
in carcinoma of the stomach and uterus, in which hemorrhages are very frequent, 
quite an extensive leucocytosis, while in cancer of the esophagus a leucopenia 
may obtain. The larger and faster the tumor grows the greater will be the 
degree of leucocytosis, a condition which is the reverse of that usually found in 
the case of the red cells. The leucocytosis of cancer is usually of the poly- 
nuclear type, but this may not be over 45 per cent. , in which cases the lymphocytes 
are relatively increased. The eosinophiles are rarely as much diminished as 
in other conditions, but they are not always increased. Myelocytes are, 
perhaps, more frequently found in cancer than in the other types of anemia, 
excepting pernicious anemia and leukemia. 

The degree of cachexia is often very extensive in cancer, but is not always 
so closely related as one would suppose to the changes in the blood. In those 
cases in which the cachexia is due to a combination of malnutrition with intoxi- 
cation by the malignant toxin the blood changes are naturally very severe, but, as 
previously stated, the more fulminating types of cancer are not associated either 
with great cachexia or with severe changes in the blood. Cachexia, therefore, 
seems to be more a function of the chronicity of cancer than of its malignancy. 

The changes in the blood in the numerous specific diseases of various 
organs show nothing characteristic in themselves. It is to be expected that in 
all chronic diseases of whatever organ, a slight anemia may be present owing to 
the effects of such disorders upon the general metabolism. These changes are, 
however, of the general type of simple anemia and are usually rapidly remedied 
by the application of the ordinary therapeutic agents. It is true that disease 
of the general organs causes changes in the composition of the fluid portions 
of the blood, which may bring about a secondary change in the cellular content. 

(e). Effects of Splenectomy. 

The effects of splenectomy are usually the combined results of severe 
hemorrhage, a preexisting anemia, of the loss of functions of this organ, and 
of intravenous infusion 1 which has been performed following the operation 

1 See Ottenberg and Kaliski, Jour. Am. Med. Assn., 1913, LXI, 213S; Nobel and Steine- 
bach, Ztschr. f. Kinderhkde., 1914, XII, 75. 



THE BLOOD. 561 

(Ewing). In comparatively healthy subjects splenectomy has often been 
performed without affecting the blood more than does any other abdominal 
operation. Immediately after removal of the spleen an increase in the blood 
pigment and a polycythemia occur (which are probably due to diminished 
hemolysis in the absence of the spleen) providing there is an adequate supply 
of iron in the diet. The most marked changes in the blood are seen in those 
cases in which the organ has been removed for rupture or idiopathic enlarge- 
ment, the loss of blood and the shock of operation giving rise to a considerable 
degree of secondary anemia. 

The red cells are frequently restored to normal in one to three months, 
due to hyper- activity of the bone-marrow, but in some cases which progress less 
favorably the anemia may be more persistent. The restoration of hemoglobin 
does not take place, as a rule, as rapidly as does that of the red cells. Following 
the operation we usually observe a polynuclear leucocytosis which may run, as 
in one case observed by the writer, as high as 75,000 cells. This leucocytosis 
usually lasts from one to two months, but may persist for several months, in 
which case the polynuclear cells are replaced by lymphocytes. Eosinophilia 
usually develops early and has been observed in some cases two or three years 
after operation. In some cases, especially those suffering from extensive 
hemorrhage, a very profound anemia characterized by great diminution in the 
number of red cells, the presence of polychromatophilic and degenerated cells, 
nucleated red cells, and a high degree of leucocytosis is observed. The leuco- 
cytes in these cases may take on the picture of an acute leukemia, but this con- 
dition is transitory as the blood improves more or less rapidly. A leucocy- 
tosis or permanent lymphocytosis are probably the only specific effects of 
splenectomy. l 

V. Parasitology of the Blood. 

(1). Malaria (Paludism; Hemamebiasis) . 

Malaria is a disease caused by the entrance of an animal parasite into 
the blood and its development within the red blood-corpuscle. This parasite 
was first studied by Laveran and belongs to the class of sporozoa. It was 
not until the recent work, especially of Grassi, Ross, and Nuttall, that we 
were enlightened as to the source of this invader. It is at present well estab- 
lished that the malarial parasite runs its sexual cycle (sporogone) within the 
body of the anopheles mosquito (Anopheles maculipennis). 2 The old idea 
that malaria was an air-borne disease, the contagion arising from stagnant pools 
in swampy regions must now be replaced by the modern mosquito theory. It 
is true that the anopheles lays its eggs upon the surface of almost stagnant 
water and that the larvae hatch in these places. The eggs are boat-like in 

1 See Musser, Arch. Int. Med., 1912, IX, 592; Giffin, Am. Jour. Med. Sc, 1913, CXLV, 781; 
Sollenberger, Biochem. Ztschr., 1913, LV, 13; Musser and Krumbhaar, Jour. Exper. Med., 
1913, XYin, 487; Pearce and Pepper, Jour. Exper. Med., 1914, XX, 19; Krumbhaar and 
Musser, Ibid., 108; Austin and Pearce, Ibid., 122; King, Arch. Int. Med., 1914, XIV, 145; 
Morris, Ibid., 1915, XV, 514. 

2 Different species of the Anophelinae vary in their ability to transmit malaria. While 
more than 100 species are known, many have not been studied. See Walker and Barber, Phil. 
Jour. Sc, B, 1914, LX, 381; also, Ludlow, Disease-bearing Mosquitos, Bull. 4. War Dept.. 
Office of the Surgeon General, 1914. 

36 



562 DIAGNOSTIC METHODS. 

ehape (each separate, the groups being arranged in ribbons) and float 
upon the surface, while the larvae He just below the surface and are in a plane 
parallel with it. These facts have led to the adoption of the modern methods 
of prevention of malaria by covering the surface of such stagnant pools with oil, 
which prevents access of air to the larvae and, in consequence, causes death. 1 

In a discussion of malaria it must be remembered that there are several 
types of this disease, depending on (1) the kind of parasite causing the infection 
and (2) the period at which the various groups of the same parasite run their 
asexual course in the host. We have, therefore, to discuss the three types 
of infecting organism, each of which is a protozoan form and is found in the red 
cells. The cycle of development of the tertian organism is approximately 
48 hours, so that with a single infection paroxysms will occur on alternate days. 
With the quartan organism the cycle of development requires 72 hours, while 
with the estivo-autumnal form it is variable, running from 24 to 72 hours. 
It is, of course, possible to have infection with more than one form of parasite 
or with more than one series of the same parasite, so that we may have daily 
exacerbations through infection with any one of these three types of parasite. 

In an examination of the blood for the malarial parasite a study of the 
fresh specimen is always desirable when possible, as the peculiar ameboid 
movements of the parasite as well as the rapid oscillatory movements of its 
granules can, of course, not be seen in the fixed specimen. Moreover, the 
peculiar brassy tone of the red cell and the irregularity in shape and size of these 
cells may best be studied in the fresh specimen. The beginner, however, will 
find that a stained specimen will yield much more definite results, providing his 
staining technic is good than will a study of the fresh specimen, as the slight 
refractility both of the cell and of the parasite in the fresh specimen makes it 
difficult in every case to get the proper illumination of the specimen. In the 
hands of an expert the examination of fresh blood is practically all that is 
required for a diagnosis in the average case, and when the parasites are moderately 
numerous the beginner can scarcely make a mistake. It would seem, therefore, 
inadvisable to rest a diagnosis upon an examination of the fresh specimen in 
cases in which no organisms are found, but to control this examination by a 
careful study of a stained specimen in which one may frequently be surprised 
at the number of parasites to be seen, although a negative result has been 
observed in the unstained specimen. A word .of caution, however, is necessary 
at this point. Frequently one observes in stained specimens many artefacts 
due to deposition of staining pigments upon the red cell, while in the fresh 
specimen areas of coagulation necrosis are not infrequently seen, so that 
the untrained observer may assume the presence of malarial organisms. For 
an absolute diagnosis of malaria it is necessary to find intracellular organisms, 
and not to be content with a single examination in doubtful cases. 2 

1 See McCoy, Pub. Health Rep., 1912, XXVII, 1029; also Craig, The Prophylaxis of Mala- 
ria, Bull. 6, War Dept., Office of Surgeon General, 1914; von Ezdorf, Pub. Health Rep., 1913, 
XXVIII, 2830; Ibid., 1914, XXIX, 503, 613, 871, 1073 and 1289. 

2 Bass has recently succeeded in cultivating the various types of malarial plasmodia. See 
Bass, Jour. Am. Med. Assn., 1911, LVTI, 1534; Bass and Johns, Jour. Exper. Med., 1912, 
XVI, 567; Lavinder, Jour. Am. Med. Assn., 19 13, LX, 42. The parasites are cultivated only 
in the red cells of human blood and are destroyed by the leucocytes as well as by the serum. 



PLATE XXVIII. 



K 



10 11 



% 



13 



16 
15/ fc ^ 



The Tertian Parasite. 



i. Normal erythrocyte. 

2, 3, 4, 5. Intracellular hyaline forms. 

6, 7. Young pigmented intracellular forms. In 6 two distinct parasites inhabit the ery- 
throcyte, the larger one being actively ameboid, as evidenced by the long tentacular 
process trailing from the main body of the organism. This ameboid tendency is 
still better illustrated in 7, by the ribbon-like design formed by the parasite. Note 
the delicacy of the pigment granules, and their tendency toward peripheral arrange- 
ment in 6, 7, and 8. 

8. Later developmental stage of 7. In 7, 8, and 9 enlargement and pallor of the infected 

erythrocyte become conspicuous. 

9. Mature intracellular pigmented parasite. 

10,11, 12. Segmenting forms. In 10 is shown the early stage of sporulation— the develop- 
ment of radial striations and peripheral indentations coincidentally with the swarm- 
ing of the pigment toward the center of the parasite. The completion of this process 
is illustrated by 11 and 12. 

13. Large swollen extracellular form. Note the coarse fused blocks of pigment. (Com- 

pare size with that of normal erythrocyte, 1.) 

14. Flagellate form. 

15. Shrunken and fragmenting extracellular forms. 

16. Vacuolation of an extracellular form. 

Note. — The original water-color drawings were made from fresh blood specimens, a, 
Leitz jVinch oil-immersion objective and 4 ocular, with a Zeiss camera-lucida, being used. 



(E. F. Faber, ./<?£.;) 
(From Da Costa's "Clinical Hematology") 



THE BLOOD. 563 

Examination of Fresh Blood. 

The technic of making a fresh specimen of suspected blood is the same 
as that previously outlined and consists in touching a perfectly clean cover- 
slip to a drop of blood and allowing the cover-slip to fall upon a clean glass 
slide. The quantity of blood should be rather small so that the red cells may 
be distinctly separated from one another. The examination is best made 
by the use of a 1/12 immersion lens. 

(a). The Tertian Organism (Hemameba vivax ; Plasmodium vivax). 

The youngest form of the tertian parasite as it appears in the red cell 
resembles very closely the spore of the parent rosette. 1 It is a small, compact, 
colorless, non-pigmented- disk (hyaline form) about 2 microns in diameter 
and shows an undulating outer rim of basophilic protoplasm which encloses 
a single large nuclear body which does not stain with methylene blue but 
shows a distinct chromatin stain with any of the modifications of the Roma- 
nowsky stain. This nuclear body is usually surrounded by a clear space which 
does not take the stain and which has been termed by Gautier "the milky zone.'' 1 
The parasite has a very rapid ameboid movement and shows a great number 
of changes in shape and position. It sometimes assumes a typical ring-like 
form which is usually a little thicker at one point, from which the name " signet 
ring" has been given. Occasionally several of these rings may be seen within 
a single blood-cell. After about 12 hours the corpuscle increases slightly in 
size, becomes somewhat paler, but still has the sharp, smooth, round outline 
of the normal cell. At this stage the ameboid powers of the organism are very 
marked, so that many pseudopodia may be seen, connected to the larger part 
of the organism by very thread-like pale and rather indistinct bands of union. 
This gives the appearance of disconnected globules of protoplasm, which 
is very slightly refractile. At this period (12 hours) pigment (hematin) appears 
in the parasite in the form of very fine light brown granules which have a very 
rapid dancing motion and are clustered especially at the ends of the pseudo- 
podia. The organism continues to increase in size and, at the same time, the 
host becomes somewhat larger, paler, but still round in outline. At the end of 
24 hours the organism fills about one-third of the cell, is still ameboid and shows 

See, also, Sinton, Ann. Trop. Med. and Parasitol., 1912, VI, 371; and Olpp, Munch, med. 
Wchnschr., 1912, LIX, 2623. Thomson and Thomson (Proc. Roy. Soc. London, 1913, 
LXXXVII, 77, and Ann. Trop. Med. and Parasitol., 1913, VII, 153) show that cultures of 
benign tertian parasites differ from those of the malignant tertian forms in that there is no 
tendency to clumping of the former types. This may explain why we do not find any but the 
young forms of the malignant tertian organism in the peripheral blood, as the clumping causes 
the larger older forms to collect in the internal organs. Ziemann, Centralbl. f. Bakteriol., 
1 Abt., Orig., 1913, LXVII, 482; Sergent, Beguet and Plantier, Compt. rend. Soc. de biol., 
1913, LXXV, 324; Bass, Am. Jour. Trop. Dis. and Prev. Med., 1914, I, 539. Miller (Jour. 
Am. Med. Assn., 1914, LXII, 1549) reports the cultivation and development of a second gen- 
eration of parasites without the removal of the leucocytes. 

1 Stephens (Proc. Roy. Soc. London (B), 1914, LXXXVII, 375, and Ann. Trop. Med. and 
Parasitol., 1914, VIII, 119) reports the discovery of a new "malarial parasite, which he names 
Plasmodium tenue. Craig (Jour. Parasitol., 1 914, I, 85) believes this to be nothing more than 
a rather atypical form of Plasmodium vivax in the unpigmented stage of development. 

Emin (Bull. Soc. Path. Exot., 1914, VII, 385) describes a further variety of the tertian organ- 
ism to which he gives the name Plasmodium vivax variety minuta. Craig does not believe the 
evidence sufficient to give this parasite specific rank. 



564 DIAGNOSTIC METHODS. 

increased pigment, which is somewhat darker in color and is less actively 
motile, being distributed throughout the substance of the parasite. In this 
form one may occasionally see the nucleus as a globular body at the end of a 
pseudopod. In the last half of the cycle of development of the tertian organism 
the growth is much more rapid than in the first half, the parasite being 
fully developed within 40 hours. The cell at this time is about one and a half 
times its normal size and is so little refractile that its outline can scarcely be 
seen. The organism is from 8 to 10 microns in diameter, is round, and is 
even less refractile than is the corpuscle. The pigment is much more 
abundant at this time and is still evenly distributed throughout the organism. 
The next stage in the development of the organism is known as the pre- 
segmenter stage. The cell becomes practically invisible, the pigment collects 
in one or more irregular clumps throughout the organism, the granules moving 
in irregular lines. At this time the periphery of the organism shows slight 
crenation and refractive dots appear irregularly in the periphery of the organism. 
The line of demarcation between the presegmenter and the segmenter is 
very slight. The corpuscle is now practically eliminated and the organism 
becomes more dense and highly refractile. The refractive dots which were 
visible in the presegmenter stage are now seen to be in the center of lines 
of separation which pass from the irregular crenated border down toward 
the center of the organism, thus marking off future segments, which are as 
a rule from 15 to 20 in number. As development proceeds the segments 
become more sharply defined until the clumps form into discrete circular 
masses with a distinctly refractile spot in the center. The pigment in these 
segment forms seems to be left in masses between the segments without any 
definite arrangement. Each segment now splits off from the mother segmenter 
cell and becomes free in the blood in the form of the original hyaline type which 
becomes attached to a red cell and soon enters it to pass through the various 
stages discussed. It is to be remembered that the hyaline forms do not modify 
their host, either in shape, color, or size, such changes being observed only at 
the time when the pigment first becomes evident. 

The preceding is a concise description of the cycle of development within 
the cell {asexual generation or schizogone), but we occasionally find tertian forms 
other than hyaline which are extracellular. These seem to be of two types, the 
degeneration forms and the gametocytes, cells capable of sexual development. 
The degeneration forms or. as they are sometimes called, extruded intracellulars, 
are sometimes the only ones seen in the specimen. These are parasites which 
have passed from the cell and have died, the organism sometimes appearing 
as if it had passed out through a very fine hole. If it be entirely extruded, the 
hemoglobin leaves the cell with it and only a shadow of the red corpuscle remains 
behind. However, this does not always occur, so that we meet with typical 
dumb-bell-shaped organisms in the plasma. If the blood be observed while 
this process of extrusion is going on, the pigment will still be extremely active 
but it gradually becomes quiet as the organism dies. The organism may 
break up into fragments forming several pigmented spherical masses or it may 
become deformed and vacuolated, constituting the so-called "sporulating" 



PLATE XXIX. 



3 4 5 6 



^ .;">- k ® * 

7 8 9 10 11 



4**N f * , 



The Quartan Parasite. 



i. Normal erythrocyte. 

2. Intracellular hyaline form. 

3. Young pigmented intracellular form. Note the coarseness, dark color, and scantiness 

of the pigment granules. 

4. 5, 6, 7. Later developmental stages of 3. Note the peripheral distribution of the pigment 

in all the parasites from 3 to 8. (Compare size and color of the erythrocytes in 5, 6, 
and 7 with 7, 8, and 9, Plate VI.) 

8. Mature intracellular form. Note that the stroma of the erythrocyte is no longer 

demonstrable. 

9, 10, 11. Segmenting forms. In 9 are shown the characteristic radiating lines of pigment. 

(Compare with 10, 11, and 12, Plate VI, and with 10, 11, and 12, Plate VIII.) 

12. Large swollen extracellular form. (Compare with 13, Plate VI.) 

13. Flagellate form. (Compare with 14, Plate VI.) 

14. Vacuolation of an extracellular form. 

(E. F. Faber,/<?c.) 
(From Da Costa's "Clinical Hematology."} 



THE BLOOD. 565 

forms. The gametocytes are found at all times in the blood after the infection 
has been established for a few days. These are not in reality extracellular 
forms as one sees them in the stained specimens surrounded by the shell of the 
corpuscle. These gametocytes are of two forms, the macrogamete or female 
cell and the micro gamete, the male cell, which is one flagellum of the micro- 
gametocyte (parent male cell). The macrogametes are large organisms, pale, 
indistinct, and three or four times as large as the red cell. Some of them show 
no trace of the corpuscle; their pigment is abundant and exhibits very active 
movements. Their nucleus is about three and one-half microns in diameter and 
is sometimes seen in the fresh specimen; either its outline is distinct or its size 
and shape may be recognized, as it is the only portion of the parasite which 
is not invaded by the pigment granules. The function of these macrogametes 
seems to be to continue the life of the organism within the mosquito after the 
organism has become fertilized by the male element or microgamete. The 
microgametocyte is smaller than the macrogamete, being eight to ten microns 
in diameter. Its pigment is very active, but soon forms a circle around the center 
and becomes stationary. Occasionally this pigment may become even more 
active than before, the margin of the cell may undulate and several flagella 
protrude. These flagella are the microgametes and are two to three times 
the length of a red blood-cell and often contain pigment granules which enable 
them to be followed when they break loose from the parent cell. After these 
flagella separate, the parent cell is seen as a small cell with central motionless 
pigment. This process of flagellation is not seen in the fresh specimen, but 
occurs 15 or 20 minutes after the blood has been drawn, which would point to 
the fact that such a process does not occur within the body. Normally, this 
change takes place only in the stomach of the mosquito. 

(b). The Quartan Organism (Hemameba malariae; Plasmodium 
malariae). 

This organism is much more rare than is the tertian. Its cycle of develop- 
ment requires 72 hours so that the normal paroxysm occurs every fourth day. 
If two groups are causing the infection there will be two days with paroxysms, 
one free day, and then two days of paroxysm following. If more than two 
groups are introduced we may have daily chills and fever, but only when the 
groups are large enough in number to cause a paroxysm. According to Ross, 
250,000,000 organisms are necessary before a chill follows. The small hyaline 
forms of the quartan parasite are not distinguishable in the early stages from 
those of the tertian type, but are easily recognized at the time pigment appears, 
as the granules of the former are coarser, darker in color, and not so actively 
motile. As the parasite grows in size the corpuscle becomes smaller and stunted 
with an irregular crenated margin. The protoplasm of the organism is more 
refractile than that of the tertian organism and hence the outlines of the pseudo- 
podia are more easily seen, although the parasite is less actively motile. In 24 
hours the red cell is quite small, crenated, and distinctly brassy in color. The 
organism is round or oval, quite distinct, slightly ameboid, and its pigment 
blackish-brown in color and gathered at the periphery, especially on one side, 



566 DIAGNOSTIC METHODS. 

thus differing from the tertian organism in which the pigment is scattered 
throughout the organism. The pigment granules have practically no motion 
at this stage of the development. As development proceeds the parasite 
fills from one-third to one-half of the cell, becomes rounder, and loses its ameboid 
power. The protoplasm is very distinct and highly refractile. During the 
third day only a rim of the cell is left and this usually takes on a dark, brassy 
tone. The organism is at this time full grown and is about seven microns in 
diameter. The pigment now passes from the periphery of the organism 
toward the center in definite radial lines, giving a wheel-like formation, with the 
pigment granules forming the spokes. Later the pigment collects in the center 
and we have the formation of the presegmenter form. Following this the 
organism becomes opaque, refractive dots appear in a single regular circle 
about the periphery, and crenations of the border appear with these dots as a 
center. Lines of division start from these crenations and run to the center, 
forming from six to twelve segments, like the petals of a flower, giving rise to 
the name " daisy," " marguerite," or " rosette" form. These quartan segments 
are much more perfect than are the tertian forms, and later separate to form 
the hyaline types which take up the development as outlined above. This 
whole cycle of the development of the quartan organism takes place in the 
peripheral blood as it does in the tertian organism, but the number of segmenter 
forms is much more numerous in the quartan type than it is in the tertian form. 
This is probably due to the fact that a large number of the tertian forms accumu- 
late in the internal organs. 

The gamete forms are not as frequently seen as are those of the tertian 
organism. They are similar in appearance, but somewhat smaller than those 
of the tertian organism and give rise to flagellation in the same manner. The 
extracellular forms are occasionally found, but not so frequently as those of the 
tertian form. 

The distinguishing marks between these two types of organisms may 
be summarized as follows: The cycle of development of the tertian organism 
is 48 hours, while that of the quartan is 72. The quartan organism is smaller, 
more refractile, less ameboid, and its pigment is coarser, darker, less motile, 
and more peripheral in position. The corpuscle infected by the quartan 
organism is smaller, shrunken, crenated, and more brassy. The presegmenter 
and segmenter stage are much more distinctive in the quartan than in the 
tertian type and more of the segment forms of the former are found in the 
peripheral blood, although the number of segments of the quartan type are less 
than those of the tertian parasite (Emerson). 

(c). The Estivo -autumnal Parasite (Plasmodium precox; Plasmodium 
falciparum) . 

This is the most dangerous type of malarial infection. The duration 
of the cycle of development varies from 24 to 72 hours. In infection with 
this organism the members of the same group do not always develop in the 
same unity, so that we may find at times an intermittent fever, but one which 
becomes more and more continuous. The hyaline forms are similar to those 



PLATE XXX. 



14 



15 16 17 



4 6 ** % 

19 20 21 22 



^ '"■■• * £* 

24 9C 
23 26 

25 



The EstivoAutumnal Parasite. 



i. Normal erythrocyte. 
2, 3. Young hyaline ring-forms. 

4, 5, 6. Intracellular hyaline forms. In 4 the parasite appears as an irregularly shaped disc 
with a thinned-out central area. In 5 and 6 its ameboid properties are obvious. 

7. Young pigmented intracellular form. Note the extreme delicacy and small number of 

the pigment granules. (Compare with 6, Plate VI, and with 3, Plate VII.) 

8, 9. Later developmental stages of 7. 
io, 11, 12. Segmenting forms. 

13, 14. Crescentic forms at early stages of their development. 

15, 16, 17, 18, 19. Crescentic forms. In 15 and 19 a distinct " bib " of the erythrocyte is visible. 
Vacuolation of a crescent is shown in 18, and polar arrangement of the pigment in 17. 
20. Oval form. 
21,22. Spherical forms. 

23. Flagellate form. 

24. Vacuolation and deformity of a spherical form. 

25. Vacuolated leucocyte apparently enclosing a dwarfed and shrunken crescent. 

26. Remains of a shrunken spherical form. 



(E. F. Faber, fee.) 
(From Da Costa's "Clinical Hematology.") 



THE BLOOD. 567 

of the tertian and the quartan types, but are slightly smaller and assume the 
"signet-ring" form much more commonly and maintain it longer. This 
early form of the estivo-autumnal parasite is distinguishable from the tertian 
by the shrinkage of the red blood-cell and from the quartan parasite by the 
smaller dimensions. In some cases the rings do not show the thickening of 
one segment, but remain of a uniform fine caliber throughout. These rings 
which do not show the distinct " signet-ring" type nearly always present two 
nuclear bodies lying at opposite poles or close together. Occasionally these 
rings appear as if unfolded and stretched across the cell like a thread, the 
nuclei appearing at irregular intervals. These rings may at times lose their 
refractility and become ameboid. As the parasite develops, a slight amount 
of pigment appears, usually seen as one or two granules which are motionless, 
as a rule, and are located at the periphery of the parasite or at the inner edge 
of the biconcavity. The cell is very commonly much shrunken, crenated, 
and brassy, even in the early stages. Some cells, which do not contain parasites, 
show the same injurious effects of the organism. The parasite at this time 
occupies about one-fifth of the cell. The infected cells now usually disappear 
from the circulation and continue their development in the lymph-glands, 
especially in the spleen. In some cases, however, the parasite does continue 
its development in the peripheral blood, but this is rare. In such blood or in 
that obtained from the spleen, the pigment appears much increased and seems 
to be rather coarse and dark in color, thus resembling very closely the quartan 
organism at this stage. It seems to be a general rule that the more malignant 
the type of estivo-autumnal malaria, the fewer older forms are seen in the 
peripheral blood, although numerous young parasites are present. In some 
cases the hemoglobin becomes concentrated around the parasite, leaving an 
almost colorless ring at the periphery of the cell. The cycle of development 
in the internal organs seems to take place within the macrophages, which 
are best studied in the fresh specimen. The parasite develops to about 5 
microns in size, which is about half the size of the cell, and when full grown 
has its pigment all in the center, never diffusely scattered as in the tertian 
organism or peripherally located as in the quartan type. This form is rarely 
seen in the peripheral circulation. The segmenters vary in size from 2 1/2 to 
5 microns in diameter, the process of segmentation being similar to that of the 
other organisms giving rise to the formation of 15 or 16 very small segments. 

Certain characteristic forms of this type of malaria appear in the pe- 
ripheral blood from about the seventh day of infection and in the internal organs 
as early as the fifth day. These forms are known as the crescents and the ovoids. 
The crescents 1 are slightly longer than the red blood-cells and show a distinctly 
crescentic shape with rounded ends, although irregular forms are at times 
observed. They are very ref ractile and usually show a fringe of the degenerated 
red blood-cell, which is more abundant in the concavity of the crescent and 
forms the so-called "bib." The pigment is large in amount and is massed at 
the center of the crescent, occasionally in the form of a sheaf or ring. The 

1 See Thomson (Ann. Trop. Med. and Parasitol., 1914, VIII, 85) for a discussion of the 
origin and development of these crescents. 



568 DIAGNOSTIC METHODS. 

granules are usually coarse and rod- shaped. These crescents very frequently 
change their shapes, becoming oval, dumbbell-shaped, or circular and then 
may resume their original crescentic form. In the circular types no trace of 
the corpuscles is seen and the protoplasm is not as distinctly refractive as is 
that of the crescent. In this form of malaria we also find pigmented leucocytes, 
both the polynuclear neutrophiles and the large mononuclears assuming this 
function. In these phagocytic cells one may see masses of pigment or even 
parasites, especially the segmenting and flagellating forms. These pigmented 
cells are also seen in the other form of malaria, but only just after the chill, while 
in the estivo-autumnal form they may occur at any time during the infection. 

Examination of Stained Specimens. 

The technic of making preparations of malarial blood for staining is 
practically the same as that outlined previously. Precaution must be taken 
to make thin smears so that the parasite may be brought out more clearly. 
The stains to be used will depend largely on the experience of the worker, but 
the writer would recommend the thionin and Nocht stains above the others, 
although the Wright and Giemsa stains will frequently give beautiful pictures. 
Stains which have been kept for some time are not always reliable, so that it is 
well to have fresh specimens of the stain on hand for use. If the blood has been 
kept for some time before staining a diffuse plasma staining with methylene blue 
will be observed. 

The Tertain Parasite. 

The young hyaline form consists of a mass of blue protoplasm usually 
grouped in ring-form with a mass of reddish-violet stained chromatin, usually 
situated at the thinner portion of the ring and extending for a large part within 
the clear achromatic or vesicular part of the parasite. These hyalines are 
2 to 3 microns in diameter. There is some discussion as to what portion of 
the parasite the name nucleus should be applied. Some give this term only 
to the chromatin staining part, while others include both the chromatin and 
achromatic portion. However this may be, it is necessary for the recognition 
of the tertian organism that the blue protoplasm and the red chromatin be both 
observed. There are frequently artefacts in the blood which resemble very 
closely these hyaline forms so that the worker must be constantly on his guard. 
Such artefacts are the Maragliano degenerations so commonly seen in red blood- 
cells and also the cases in which blood-platelets lie upon a red blood-cell. 
Any structure which lies upon a red blood-corpuscle appears surrounded by a 
colorless zone, while the true malarial ring is in direct contact with the hemoglobin 
of the cell. Moreover, such artefacts will not show the chromatin staining 
portion which is so characteristic of the hyaline ring. In the specimens examined 
at the end of 24 hours, one will observe that the achromatic area has become 
somewhat larger, while the chromatin portion seems to be grouped in more 
irregular masses, some cells appearing to have several nuclei. In the full- 
grown parasite the chromatin breaks up into a cluster of fine granules which are 
scattered diffusely through the cell in the form of strands and masses. These 
chromatin clumps separate into from 15 to 20 dense round masses, around 



PLATE XXXI. 




Tertian Malarial Parasite. (Wright's Stain.) 



THE BLOOD. 569 

which the protoplasm collects with them as a center. The protoplasm at this 
stage is distinctly achromatic and is always so in the segmenting cell. The 
distinct, achromatic, milky zone surrounds each segmentary chromatin clump, 
while the general protoplasm shows a diffuse faintly basic staining. The 
pigment which must not be confused with the chromatin is pushed toward the 
periphery and, after segmentation is complete, collects in masses near the 
center. It is to be recalled that at the time the pigment collects in the center 
in the fresh specimen there is no distinct evidence of segmentation, although 
this segmentation shows quite distinctly in the stained specimen. 

The sexual development of the parasite is easily followed in some cases 
in the stained specimen. According to Stephens and Christophers, the young 
gamete is characterized by the position of the chromatin as it lies in the center 
of the vacuole instead of at the edge. During the development the gamete 
is occasionally filled with basophile particles which are known as Plehn's karyo- 
chromatophilic granules or Schugner's granules. The full-grown macrogamete 
contains an abundance of protoplasm which stains a deep blue and a small 
amount of chromatin in a compact mass, which is peripherally placed and 
surrounded by a thin vacuole-like area. The pigment of these female cells 
is uniformly distributed throughout the cell and the inclosing red blood-cell 
can be seen only with difficulty. The chromatin is much more voluminous 
in the microgametocytes, but is looser and centrally placed in a large achromatic 
zone arranged in the form of a band which stretches clear across the cell. The 
protoplasm is in the form of a ring around the nucleus and stains more of a 
grayish-green color than does the bluish protoplasm of the macrogamete. 

The Quartan Parasite. 

The structure of the quartan parasite resembles very closely that of the 
tertian form, but in the hyaline type the chromatin mass is less distinct and is 
in the form of an irregular clump of granules in the older forms, while in the 
younger a cluster of fine granules without any distinct achromatic zone is seen. 
As development proceeds the parasite generally takes a form extending across 
the cell and usually occupies the larger portion of the red cell which has become 
shrunken and irregular in shape. The segment forms are much more distinct 
in the quartan type than in the tertian and show much more regular and 
geometric lines of cleavage with the chromatin exactly in the center of the cre- 
nated surface. The pigment granules are coarser and much more distinct 
than in the tertian form and are more peripherally located. 

The Estivo-autumnal Parasite. 

The hyaline forms in this type show the chromatin in two or more masses 
or filaments. The protoplasm is scantier than in the other forms and remains 
so throughout the cycle of development of this parasite. A very characteristic 
appearance of the hyaline rings of this type seems to be the thickening of the 
protoplasmic layer opposite the chromatin mass. The gamete forms are 
distinctly spherical, being of the same thickness all the way round. Their 
nucleus forms a portion of the ring, but does not project as in the schizonts 
(the asexual parasites). The red blood-cells in these sexual types usually show 



570 



DIAGNOSTIC METHODS. 



no coarse granular stippling. The crescent forms, which are characteristic of 
this type of malaria, show the chromatin in a loose network which occupies 
the larger portion of the cell, has little blue staining protoplasm, and has its 
pigment scattered throughout its body. This is the male form and is somewhat 
kidney-shaped and is shorter and broader than the female type. The female 
crescent is longer and narrower, its chromatin more or less compact and 
centrally located, its pigment in a ring around the nucleus or in a clump at 
the center, while its protoplasm is more or less extensive and takes a distinct 
bluish tinge. We also find two types of the circular form. The microga- 
metocyte is smaller than the red cell, distinctly spherical in shape, with its chro- 
matin in the center in a large irregular mass, or in several dense masses near the 
periphery. These masses containing chromatin material are later extruded and 
form the flagella or microgametes. The macrogamete is two or three times 
the size of the microgametocyte, is often of a triangular shape, and has abundant 
blue-staining protoplasm. The chromatin is in a single mass at the periphery 
and is surrounded by a circle of pigment. 

The examination of the stained specimens does not give as great an 
opportunity for study of the developmental cycle of these parasites as does the 
examination of a fresh specimen. The conditions found in the fresh blood 
resemble more nearly those found in the stomach of the mosquito than in 
the circulating blood, so that many pictures seen in the fresh specimen are 
practically never found in the stained slide. 1 It is further to be said that we 
do not always find malarial organisms either in the fresh or stained specimen, 
although the patient may be at the time suffering from malaria. It may be 
stated, as a rule, that in all well-marked initial attacks of malarial fever the 
parasite may be found in the blood if it be examined within 18 hours of the 
chill. The energetic use of quinin has so much influence upon the ameboid 
types of the parasites, that the blood may fail to show any of these organisms, 
although the patient may die from the effect of the infection. It is safe, how- 
ever, to state that there is practically no case of malaria in which parasites may 
not be found in the blood, if frequent and repeated examinations are made. 

Development of the Organism within the Mosquito (Sporogony). 

The cycle of development of the malarial organism has been more closely 
followed in the mosquito in the case of the estivo-autumnal parasite. For 
any development to occur within the body of the mosquito it is necessary 
that the macrogamete become fertilized, so that the course in the mosquito 
is one of sexual development. The microgametocytes throw out their flagella 
(microgametes) and the macrogamete ripens in the stomach of the mosquito by 
casting off karyosomes (polar bodies consisting of chromatin), and in so doing 
causes the formation of a slight mound at one portion of the organism through 
which the free flagellum enters. This process occurs in from one to one and 
a half hours after the mosquito has bitten a patient infected with malaria. The 
nuclear material of the macrogamete and microgamete then unite. The cell 
then forms a distinct motile spindle shape called the vermiculus or obkinet. The 

1 See Clark (Jour. Exper. Med., 1915, XXII, 427) for a discussion of the value of the study 
of placental blood films in the diagnosis of malaria. 



PLATE XXXII. 




ESTIVO-AUTUMNAL PARASITE. (WRIGHT'S STAIN.) 



THE BLOOD. 



571 




Fig. 144. — Cycles of the Malarial Parasite. {Deguy et Guittaumin.) 
I, Beginning of the development of the parasite as the ameboid intra-cellular body. 
/, II, III, and IV represent the endoglobular cycle or schizogony. I, II, II', II", III", 
and IIP represent the extra-cellular cycle or sporogony . In IF, there are two free gametes 
(g), one microgametocyte (m), one microgamete (m'), and the union of a macrogamete 
and a microgamete (ra"). Ill', free gametes; III", fertilized macrogamete, taken up by 
the mosquito, A. In B, C, D and E it becomes encysted in the gastric musculature forming 
the zygotes (z and z'). In E, the sporozoits are formed and in F they are thrown out by 
the saliva of the mosquito. In G, these are free to enter the cell forming the ameboid bodies. 



572 DIAGNOSTIC METHODS. 

size of this fertilized macrogamete is from 20 microns up and may be found in 
about 48 hours after the blood has been ingested. This motile form is found 
only in the stomach of the mosquito. The vermiculus then bores its way 
through the epithelial cells of the intestinal wall and becomes encysted between 
the intestinal epithelium and the elastic layer, which forms the membrane of 
the spore cyst (zygote, oocyst, sporoblast). This zygote increases rapidly in 
size and the nucleus divides rapidly. In its growth it bulges outward from 
the intestinal wall forming pendulous tumors into the body cavity which growth 
may vary from four and a half to ninety microns in diameter. This stage 
is associated with the appearance of much pigment and is called the medium 
zygote or medium sporoblast stage. The protoplasm gathers around the 
divided nuclei, forming daughter cysts which are connected by bridges of 
protoplasm forming the stage known as large zygote, large sporoblast, or large 
oocyst. In each of these divisions the nucleus divides many times, the daughter 
nuclei remaining on the surface of each daughter cyst. The protoplasm now 
collects around each daughter nucleus, the first forming spherical cells which 
then elongate into threads lying parallel over the remains of the sporoblasts. 
These threads are called sporozoits and have an elongated nucleus. The 
final length of these sporozoits is about 14 microns and their width about 
1. Their protoplasm is thick, homogeneous, and very refractive. They 
are sometimes present to the number of 10,000 in some zygotes, but more 
frequently are not so numerous. As the oocyst becomes larger it bursts into 
the body cavity, the sporozoits of each cyst ripening at about the same time. 
These sporozoits wander at first free, but soon collect in the salivary gland of 
the mosquito. They are motile and move with a bending and gliding motion. 
When they are inoculated into the blood of man by the bite of the female 
mosquito they attach themselves to the red blood-corpuscle and finally penetrate 
it to form the initial hyaline type of the organism. The period of incubation 
after the bite of the mosquito is usually between the eighth and twelfth day, 
when the first chill will appear, although the exact time of appearance of the 
initial symptoms will depend upon the number of sporozoits introduced into 
the circulation (Emerson). The anopheles is the only type of mosquito 
which is at present known to be the host of the malarial organism and to 
give rise to the development of the gameto-schizonts (the sexual cells), which 
the bite of the female animal introduces into the blood cells which are known 
in their future development as schizogones. The sexual cycle within the mos- 
quito is known as the sporogone, which Rowley-Lawson 1 has recently shown 
may occur in the circulating blood of the human host. 

General Changes in the Blood in Malaria. 

There are few conditions which lead so rapidly to such an extreme reduction 
in the red cells as does acute malaria. 2 An acute attack may reduce the red 

1 Jour. Exper. Med., 1911, XIII, 263; Ibid., 1913, XVII, 324. 

2 Rowley-Lawson (Arch. Int. Med., 1912, IX, 420) believes that this severe anemia is due 
to the migration of the plasmodia from corpuscle to corpuscle. See James, Jour. Infect. 
Dis., 1913, XII, 277; Brown, Arch. Int. Med., 1913, XII, 315; Jour. Exper. Med., 1913, 
XVIII, 96; Rowley-Lawson (Ibid., 1914, XIX, 450 and 523; Ibid., 1915, XXI, 584) discusses 
the extra-cellular relation of the parasite to the red corpuscle. 



THE BLOOD. 573 

cells to as low as 500,000 cells as reported by Kelsch. Frequently a reduction 
of 1,000,000 is observed during the first day, with a progressive reduction 
as the time goes on. In the afebrile period of the disease a continuous fall 
is observed, but this is much less rapid. The regeneration of the cells is 
very active so that an increase in the number of cells has been observed directly 
after an attack in some cases. In cases of chronic malaria the red cells are 
commonly reduced to as low as 583,000 (Kelsch), while when attacks occur 
only at intervals and are promptly stopped by quinin no reduction in the red 
cells may follow (Marchiafava). In cases of moderate severity the usual 
changes of secondary anemia are present in the red cells. Polychromatophilia 
and granular degeneration of the reds progresses steadily, while the hemo- 
globin content of the cells may be markedly reduced. Frequently cases are 
seen in which the anemia takes on the absolute pernicious type, so that the 
parasites seem to have been massed in the bone-marrow. As Ewing states, 
" there can be no doubt that the tendency of the estivo-autumnal parasite to be 
massed in the bone-marrow, in both ameboid and crescentic phases, and 
the excessive demand on red-cell production arising in the disease render 
pernicious malaria an extremely favorable condition for this disturbance of 
the structure of the marrow and the development of specific megaloblastic 
changes." Besides the changes which can be directly referred to anemia 
or toxemia, changes in the size of the cell are quite constant, the tertian parasite 
causing from the start swelling of the cell and progressive loss of hemoglobin, 
while the quartan and estivo-autumnal forms cause the red cell to shrink and 
take on a peculiar brassy tone. 

The leucocytes do not show very characteristic changes. In the acute 
malarial attacks of average severity the-absence of leucocytosis is of considerable 
corroborative value, although a slight leucocytosis amounting to about 10,000 
with an increase in the percentage of polynuclear cells has been observed by 
Billings and others. Except during the three or four hours immediately 
following a chill malarial blood usually shows a diminished number of leucocytes 
with a distinct relative lymphocytosis, which finding is that seen in typhoid 
fever. In the more severe estivo-autumnal attacks a definite leucocytosis 
has been distinctly observed, especially in the hemoglobinuric or black-water 
type of malarial infection. The extent of the leucocytosis varies between 10,000 
and 35,000, although many attacks fail to cause any distinct increase. During 
the afebrile periods the eosinophile cells are usually increased and may be observed 
throughout the course of the attack. Neutrophile myelocytes are occasionally 
present and rarely eosinophile myelocytes. Pigmented leucocytes are seen in 
the majority of cases, especially in the severe and fatal cases, the pigmented 
leucocytes being more closely related to the severity of the paroxysms than to 
the extent of the deposits in the various viscera. These pigmented or phagocytic 
cells include mononuclear and polynuclear leucocytes and a few endothelial 
cells. The large and small mononuclears usually contain pigment or rosettes, 
while many of the polynuclear leucocytes also contain the parasites. These 
phagocytes may contain, besides parasites and malarial pigment, hematoidin, 
hemosiderin, red blood-cells, leucocytes, and occasionally an unknown crys- 
talline pigment. 



574 DIAGNOSTIC METHODS. 

(2). Relapsing Fever (Famine Fever). 

The cause of this peculiar fever is the spirillum of Obermeier and is not 
a member of the class of bacteria but, more properly speaking, belongs to the 
class of spirochete. This organism is between 16 and 40 microns in length 
and about 1 micron in width, but is subject to considerable variation in size. 
It is thin, sharply curved, and appears to be structureless. It takes a deep 
chromatin stain and also stains with methylene blue in from two to five minutes. 
It is seen in the blood only during the febrile period of the disease and at that 
time is actively motile with a rapid wavy motion, much resembling the move- 
ments of a coiled spring in its stretching and collapsing. It moves rather 
slowly among the corpuscles, but does not disturb them to any extent. 1 Cases 
have been reported in which these spirochete are present in the blood 24 hours 




Fig. 145. — Spirillum of Obermeier. (Pitfield.) 

before the chill, but they are usually to be found in larger numbers at the 
time of the rise in temperature, increasing rapidly from day to day. The fever, 
as a rule, continues about six days, at the end of which time these parasites 
leave the blood. Strangely enough these organisms have been found in 
varying numbers in different parts of the circulation, while there does not 
seem to be any strict parallelism between their number and the height of the 
fever. Loewenthal has applied the agglutination test to the blood of suspected 
cases and finds the reaction positive in 85 per cent, of the cases in the periods 
in which the parasites are absent. Nothing of a characteristic nature is ob- 
served in the general blood picture. 

Cases of relapsing fever are practically never found in the United States, 
unless imported through the medium of emigrants from Russia and especially 
India. The cases reported by Wellman show that relapsing fever, as found 
in West Africa, may arise from the bite of a tick transmitting the spirillum 
of Obermeier. 2 

1 Noguchi (Jour. Exper. Med., 19 12, XVI, 199) has succeeded in cultivating this organism 
and shows that this pathogenicity is not thus diminished. 

2 Nicolle, Blaizot and Conseil (Ann. de l'lnst. Pasteur, 19 13, XXVII, 204) show that the 
bite of the louse does not transmit this disease as is commonly supposed. If the louse be 
crushed on the skin and there are abrasions on the skin from scratching, the parasites gain 
access to the system. 



THE BLOOD. 



575 



(3). Sleeping Sickness. 

This very interesting condition which is so prevalent in Central and 
West Africa seems to be due to an actively motile fusiform flagellate known 
as the trypanosoma Gambiense, which can be found in the blood free in the 
plasma (never intracorpuscularly), moving with a screw-like motion among 
the red cells which it does not seem to disturb. This parasite doubtless has 
a sexual development, its host being the common fly, Glossina palpalis, while 
closely related trypanosomata are transmitted by the bites of various flies, 




Fig. 146. — Trypanosoma gambiense. (Da Costa.) 

especially one of the seven varieties of the tsetse fly. This organism is from 
two to three times as long as a red blood-corpuscle (18 to 25 microns) and 2 to 
21/2 microns wide, having a flagellum anteriorly and an undulating membrane 
extending its entire length. 1 In the fresh blood specimen these parasites 
should be looked for with only a medium magnification. These parasites 
vary much in number sometimes being absent for a long period and then suddenly 
reappearing in large numbers. Symptoms of the disease seem to bear 
little relation to the number of parasites in the peripheral blood, so that in 
some cases it may be necessary to examine the fluid in the edematous areas 
or even to puncture the cervical lymph-glands. 2 When these parasites are 
stained with a polychrome dye they show a rather large red nucleus about the 
middle, a centrosome staining intensely in a vacuole-like area near the blunt 
posterior end, and a line of chromatin taking a dense red stain running down 
the edge of the undulating membrane and terminating in the flagellum which 
is also stained red. The protoplasm of the body takes a distinct blue stain. 
The parasite contains no pigment and, therefore, obtains its nourishment 
from the plasma and not from the red cell. 

1 See Thomson and Sinton (Ann. Trop. Med. and Parasitol., 19 12, VI, 331), who have suc- 
ceeded in cultivating this organism. 

2 Wolbach and Binger (Jour. Med. Research, 1912, XXVII, 83) show that the trypano- 
somes are not confined to the blood-vessels and lymphatics, but invade the tissues. See, also, 
Turtle, Jour. Am. Med. Assn., 1915, LXV, 240. 



576 DIAGNOSTIC METHODS. 

This disease may take an acute course, but as a rule is exceedingly chronic, 
running for years, but becoming fatal as soon as the parasite reaches the cerebro- 
spinal fluid. The true sleeping sickness appears only when the cerebrospinal 
fluid is invaded and seems to be, according to the recent work of Koch, directly 
amenable to treatment with atoxyl. In examining the cerebrospinal fluid 
for these parasites it is best gently to centrifuge the fluid for five minutes, after 
which the sediment may be examined under a vaselined cover-glass. 

There are many other types of trypanosomata, but the Gambiense form is 
the more important. This is pathogenic toward man, but cannot be distin- 
guished from the trypanosoma of the tsetse fly which is so fatal to the horse 
and mule (trypanosoma Brucei), that of the surra disease 1 (trypanosoma Evansi), 
or that of dourine (trypanosoma Equiperdum). 

(4). Kala-azar. 

Through the researches of Donovan, Leishman, and Ross, parasites 
have been demonstrated in the blood which are probably directly associated 
with the condition known as kala-azar, tropical splenomegaly, piroplasmosis, 
cachexial fever, and dum-dum fever. The organism has been called the 
Leishman- Donovan body, and is a small oval, round, or oat-shaped body from 
2 1/2 to 3 microns in diameter. These bodies have a definite cell outline 
and contain two chromatin masses, a larger one, a nucleus which is almost round 
or oval and stains faintly, and a smaller bacillus-shaped centrosome which 
stains deeply and is directed almost at right angles to the axis of the nucleus. 
These two chromatin masses are both in the long axis of the cell, the larger one 
being at the periphery. Many of these forms are vacuolated and the outline 
of the cell cannot always be seen, although these two masses thus arranged are 
distinctive. They are easily stained with the various polychrome dyes and are 
best studied with he highest lenses. These bodies probably represent a stage 
in the development of a trypanosome as shown by the work of Leishmann and 
Statham. They are not found in the circulating blood as a rule, but they have 
occasionally been reported in the form of intracellular bodies in fatal cases. 
They are easily seen in the blood obtained by splenic puncture and also in the 
granulation tissue taken from the ulcers. 2 Many are found in the mesenteric 
lymph-glands, bone-marrow, and liver. Some of these bodies lie free, but most 
of them are intracellular, either in the leucocytes, endothelial, or splenic cells, 
and frequently in large masses in the macrophages. 3 

The changes in the blood are those of a moderate anemia, associated with 
a leucopenia with a relative and absolute increase in the number of the large 
mononuclears. The average leucocyte count is about 2,000. 

1 Mitzmain (Philippine Jour. Sc, B, 1913, VIII, 223) shows that this disease is transmitted 
by the tabanid fly (Tabanus Striatus Fabricus). 

2 Bates (Jour. Am. Med. Assn., 1913, LX, 898) reports a case of Leishmaniosis of the 
nasal mucosa, the organisms being obtained from direct smears made from the ulcers. See, 
also, Patton Indian Jour. Med. Research, 1914, II, 492; Mackie, Ibid., 510. 

3 Darling (Jour. Exper. Med., 1909, XI, 515) has reported the finding of an intracellular 
parasite, the histoplasma capsulatum, as the cause of Histoplasmosis, an infectious disease 
of the Canal Zone much resembling kala-azar. The parasite is small, round or oval, 1 to 
4 fi in diameter, possesses a polymorphous chromatin nucleus, basophilic cytoplasm and 
achromatic spaces all enclosed within an achromatic refractile capsule. 



THE BLOOD. 



577 



(5). Filariasis. 

This is a condition associated with the presence of filarial in the blood 
(filaria sanguinis hominis). While many of these filarial 1 are known; the 
most common one is the filaria Bancrofti (filaria nocturna). These are from 
270 to 340 microns (0.2 to 0.3 mm.) long and from 7 to 11 microns broad. 
They are enclosed in a sheath which is considerably longer than is the parasite 
and shows fine cross striations. The anterior end of the parasite is abruptly 
rounded and has a six-tipped prepuce and a sharp fang, while the posterior end 
tapers for about two-fifths of the length of the parasite. The median axis 
of the parasite is granular. The movement of these parasites is distinctly 
progressive at first as seen under the microscope, but they soon become motion- 
less, appearing to attach themselves to the glass slide at their anterior end. 
Strangely enough these embryos appear in the circulation only toward evening, 
their numbers gradually rising to a maximum about midnight and diminishing 




Fig. 147. — Filaria Bancrofti. (Da Costa.) 



toward dawn. During the day they are found in the internal organs, especially 
the lungs. The forms appearing in the blood are practically all embryos, 
as the adult types lie in the lymphatics where they obstruct the lymph flow. 

The obstruction in the lymph-glands may also be brought about by the 
eggs, which are 25 to 38 microns long by 15 broad. The embryos reach the 
general circulation only through the thoracic duct. The female filaria is 85 
to 150 mm. long, with a distinct neck, a head with a simple, minute, terminal 
mouth, and a plain cylindrical body covered by a striated cuticle and tapering 
toward the neck and tail. The tail ends bluntly and has a small depression 

1 Wellman and Johns (Jour. Am. Med. Assn., 1912, LIX, 1531) have succeeded in culti- 
vating the hlaria immitis on artificial media. See Fulleborn, in Kolle and Wassermann's Hand- 
buch der pathogen. Mikroorg., 1913, VIII, 185; Johns and Querens, Am. Jour. Trop. Dis. 
andPrev. Med., 1914, 1, 620; Smith and Rivas, Ibid., 1914, II, 357; Dutcher and Whitmarsh, 
Ibid., 1915, III, 65; Johnson, Southern Med. Jour., 1915, VIII, 630. 

37 



578 DIAGNOSTIC METHODS. 

surrounded by two lips. The male is about 80 mm. long, without a neck and 
having a tendril-like tail rolled into one or more spirals. 

Like the malarial organisms, the filaria has an intermediate host in the 
mosquito, both of the culex and anopheles variety. The embryos, which 
are taken up by the bite of the mosquito, cast off their sheath in about one 
hour in the stomach of a mosquito. Some of these embryos die at this stage, 
but others bore actively through the intestinal wall to the muscle, where they 
remain. In the next two or three days the embryo becomes larger and its 
alimentary tract develops. On the seventh day the worm is about 1 1/2 
mm. long and is perfectly developed. It now travels toward the head and 
takes its position in the labium, whence it enters the blood of its new host 
during the biting by the insect. A large number of these adult forms is neces- 
sary to cause very severe cases and many years may pass before any symptoms 
are manifest. 

In examining the blood for the filaria, it is best to take a specimen late 
at night and to make a very thick, fresh specimen which should be examined 
with a low power. Besides the ordinary anemia which may develop in such 
cases, we find a very striking eosinophilia which may run from 4 to 17 per cent. 

A very characteristic finding in such cases is the condition of hematochyluria 
followed by chyluria. This hematochyluria seems to be due to rupture of 
the varicose lymph-vessels of the bladder, as these form a large part of the 
collateral circulation when the thoracic duct is occluded (Emerson). Such 
attacks may occur for years and be separated by long intervals. Their onset 
is spontaneous or following exertion and is usually associated with pain and 
fever. The urine shows the presence of blood, chyle (as high as 3.8 per cent, 
fat), and embryos. 

Many other forms of filariae are known, but this Bancroft type seems to 
be the more important. While this disease occurs endemically in the tropics 
there are undoubtedly many cases in this country. It is, therefore, wise in 
a case showing lymph tumor, elephantiasis, and hematochyluria, especially 
when pain and fever and enlarged spleen are present, to examine the blood 
for the filaria Bancrofti. 

(6). Syphilis. 

The search for the etiologic factor of syphilis has extended over a period 
of many years and various organisms have been described, from time to time. 
However, none of these former parasites have stood the test of criticism. 
Through the work of Schaudinn and Hoffmann in 1905, a parasite was dis- 
covered which was so constantly associated with syphilitic lesions, whether 
primary, secondary or tertiary, that it was almost universally regarded as the 
causative agent in this disease. The cultivation of this organism in pure 
culture by Noguchi (see p. 581) and the demonstration of the pathogenic prop- 
erties of this cultivated parasite have definitely established the etiologic re- 
lationship of this organism to syphilis. This organism is called the spirochete 
pallida or treponema pallidum. 

While it is now a relatively simple procedure and a matter of almost daily 



THE BLOOD. 579 

laboratory routine to demonstrate the presence of these organisms in the pri- 
mary and secondary syphilitic lesions or in the blood of affected patients, 
the detection of the spirochete in the tertiary stages or in the parasyphilitic 
conditions is not by any means always successful. It is in the latter types 
of this disease that the Wassermann and luetin tests are so frequently called 
upon to settle the diagnosis, as the primary and secondary stages are more 
amenable to direct diagnosis. 

It has, of course, been known for some time that chronic nervous conditions, 
such as general paralysis and tabes dorsalis, were closely associated with long- 
standing syphilitic infection, but this relationship remained to be clearly proven. 
Noguchi and Moore 1 have demonstrated the presence of the treponema in the 
brain tissue of paretics and also in the posterior columns of the spinal cord 
in tabes. These results have been abundantly confirmed by others. It is 
true that the results show only about 25 per cent, of positive findings in these 
cases but this is not surprising in view of the latent character of the infection. 
Such a result indicates, merely, the association of these organisms with the 
pathologic lesions of the disease and does not prove their absolute etiologic 
relationship to syphilis in general. Later work by Noguchi and others has 
clearly shown that typical syphilitic lesions may be caused in rabbits by inocu- 
lation of an emulsion of the brain of a paretic individual. The results show 
about the same percentage of successful inoculations as do those obtained 
from direct histologic examination of the tissues. 2 Graves 3 goes even a step 
further and believes that his experiments show that rabbits may be infected with 
syphilis directly from the blood of general paretics. The proof is, therefore, 
absolute that these diseases, general paralysis and tabes dorsalis, are the re- 
sult of actual syphilitic infection of the brain and spinal cord. It is evident, 
therefore, that the Wassermann and luetin tests can be of inestimable service 
in such cases in the way of clearing up a diagnosis. 

The spirochaeta pallida 4 {treponema pallidum) derives its name from its 
low refractive power and the difficulty with which it takes up anilin dyes. It 
has a very delicate structure, usually presenting 10 to 40 deep spiral incurvations 
in the larger specimens or only a few in the smaller ones. Its length varies 
between 4 and 10 microns and its width does not exceed 1/2 micron. The 
organism has been demonstrated in the circulating blood, in the scrapings 
obtained from the chancre, in the incised papules, in smears from the mucous 
patches, and in the fluid aspirated from the inguinal glands. It seems to be 
easily demonstrable in the blood from a splenic puncture, while in the congenital 
forms it is found in the internal organs and in the peripheral blood. A char- 

1 Jour. Exper. Med., 1913, XVII, 232; Noguchi, Jour. Am. Med. Assn., 1913, LXI, 85; 
Munch., med. Wchnschr., 1913, LX, 737; Jour. Cutan. Dis., 1913, XXXI, 543; Forster and 
Tomasczewski, Deutsch. med. Wchnschr., 1914, XL, 694'. 

2 See Hoche, Med. Klin., 1913, IX, 1065; Forster and Tomasczewski, Deutsch. med. 
Wchnschr., 1913, XXXIX, 1237; Wassermann, Ibid., 1281; Levaditi, Marie and Bankowski, 
Ann. de lTnst. Pasteur, 1913, XXVII, 577; Nichols and Hough, Jour. Am. Med. Assn., 1913, 
LXI, 120; Wile, Ibid., 866. 

3 Jour. Am. Med. Assn., 1913, LXI, 1504. 

4 See McDonagh, Brit. Jour. Dermatol., 1912, XXIV, 381; and Ross, Lancet, 1912, II, 
1105. 



5 8o 



DIAGNOSTIC METHODS. 



acteristic difference between this spirochaeta and some other types (spirochaeta 
buccalis), with which it might be confused, is that its ends lie above and below 
a longitudinal line drawn through the center of its curvatures, while in the other 
forms the ends lie on the projection of such a line. The organism moves in an 
oscillatory manner about its longitudinal axis, its movements being winding, 
bending and whipping, while in the spirilla the longitudinal axis remains 
rigid. Schaudinn demonstrated the existence of a flagellum at each end, while 
the other spirochaete have an undulating membrane. 




Fig. 148. — Spirochaete pallidas and refringens. (Pitfield.) 
The darker ones are the refringens. 



These organisms are seen only with great difficulty in the specimens of 
fresh blood, but thanks to the introduction of the ultra-condenser (the dark- 
field illuminator) we are in a position to see these organisms, both in the splenic 
and peripheral blood, although considerable practice is necessary properly to 
adjust the light. 1 These organisms do not take anilin dyes readily, so that 
special methods have been advanced for their demonstration in smears. A very 
good stain for them is the Goldhorn stain. The smears are fixed with pure 
methyl alcohol for 15 minutes and are then covered with the stain (polychrome 
methylene blue) for three to five seconds, when the excess is drained off. The 
specimens are then slowly introduced into clean water with the film sides down. 
Keep the slide in this position for four to five seconds and then shake in the 

1 See Sidwell and Smith, 111. Med. Jour., 1914, XXVI, 418. 



THE BLOOD. 



S8i 



water to remove the excess of the dye. The spirochete appear of a violet 
color. This violet tint may be changed to a bluish-black by covering the 
specimen with Gram's iodin solution for 15 to 20 seconds, after which it is 
washed and dried as usual and the examination made with the immersion 
lens. The writer has also found the use of the Giemsa stain very reliable, 
especially when the staining is continued for 18 hours (see Exudates). Other 
stains, such as that of Levaditi, have been advocated, but they do not seem to 
give any better results and are more complicated. For staining the spirochete 
in tissues the Levaditi stain is admirable. 




Fig. 149. — Ultra-condenser of Reichert. 

The examination of the blood is very often disappointing, owing to the fact 
that few spirochete may be present in the specimen. Better results are obtained 
by examination of specimens from a curettage which has been carried sufficiently 
far to allow serum to appear. This serous fluid is then spread upon slides 
and treated in the usual manner (see Exudates). 



Cultivation of Treponema Pallidum. 

As previously stated, the causative factor of syphilis has been definitely 
settled only by fulfilling Koch's postulates regarding pure cultures and the 
production of the disease by means of these pure cultures. Schereschewsky, 1 
Miihlens 2 and Hoffmann 3 were able to cultivate the pallidum but were unable to 
reproduce syphilitic lesions by means of their cultures. Bruckner and Galasesco 4 
and Sowada 5 reported the successful reproduction of the lesions by injection of 

1 Deutsch. med. Wchnschr., 1909, XXXV, 835, 1260 and 1652; Ibid., 1913, XXXIX, 1408. 

2 Deutsch. med. Wchnschr., 1909, XXXV, 1261; Klin. Jahrb., 1910, XXIII, 339. 
3 Ztschr. f. Hyg. u. Infectkr., 1911, LXVIII, 27. 

4 Compt. rend. Soc. de biol., 19 10, LXVIII, 684. 

s Deutsch. med. Wchnschr., 1911, XXXVII, 682; Med., Klin., 1914, X, 161. 



582 DIAGNOSTIC METHODS. 

their " young impure cultures" but, as neither investigator was able to grow a 
second generation of these so-called cultures in any medium, their results are 
questionable. Recently Noguchi 1 has succeeded not only in producing many gen- 
erations of pure cultures of the pallidum but, also, in reproducing the syphilitic 
lesions by use of these pure cultures. His work is of the greatest importance 
as it absolutely establishes the spirochaeta pallida as the etiologic factor of 
syphilis. 

His technic is as follows: As the material for obtaining the cultures 
he uses the spirochaetae-containing testicular tissue of rabbits, which have been 
inoculated with human syphilitic material. From this first generation in rabbits 
any number of generations may be derived by transmitting the strain, at appro- 
priate intervals (four to six weeks), from rabbit to rabbit. By this means the 
spirochaatae are almost free from banal bacteria. 

The culture media is the following: 16 c.c. of serum- water (3 parts dis- 
tilled water and 1 part serum of sheep, horse or rabbit) are placed in tubes 
20 cm. long and 1.5 cm. wide. These tubes are then sterilized by fractional 
sterilization at 100 C. for 15 minutes on each of three successive days. Then a 
small piece of freshly removed sterile rabbit tissue (preferably kidney or testicle, 
although heart muscle may be used but not liver) is placed in each tube which 
is then incubated at 37 C. for two days and examined for sterility. To each 
tube a layer of sterile paraffin oil is added to shield the medium from contact 
with air and to prevent evaporation. The method of cultivation must be 
strictly anaerobic in obtaining the first generation of pallida. Noguchi employs 
a combination of hydrogen gas, vacuum and pyrogallic acid in an anaerobic 
apparatus, which is fully described in his article. He believes the following 
conditions essential in obtaining his first generation: (1) the presence of suit- 
able fresh sterile tissue in serum water, (2) strict anaerobiosis, (3) a slightly 
alkaline reaction as furnished by the serum and tissue, and (4) a temperature 
of about 35 to 37 C. 

When once adapted to the artificial serum-water tissue medium, the 
pallidum grows well under less strictly anaerobic conditions. In fact, certain 
strains even grow well in a medium containing agar, provided suitable fresh 
tissue is placed low down in a high column of medium (serum-water or serum 
agar) , and is covered with paraffin oil. Out of ten strains of treponema pallidum, 
six have been cultivated in the above medium. The difficulty in this work 
seems to be to obtain the first generation. The culture may be purified by per- 
mitting it to grow through a Berkefeld filter, which the pallida pass about the 
fifth day. Certain strains grow together with bacteria along the stab canal in a 
serum-agar tissue medium. But while the bacteria do not grow out into the 
surrounding medium, the pallida grow out gradually in a pure state. The 
characteristics of this pure culture may be obtained from the literature. 2 

The morphology and motility are quite typical, difficulty being experienced 

1 Jour. Exper. Med., 1911, XIV, 99; Ibid., 1912, XV, 90; Ibid., 201; Ibid., 1912, XVI, 211. 
See, also, Nichols, Ibid. 1914, XIX, 362; Hartwell (Jour. Am. Med. Assn., 1914, LXEII, 142) 
reports the isolation of this organism from the blood. 

2 See Meirowsky ("Studien iiber die Fortpflanzung von Bakterien, Spirillen und Spiro- 
chaten," Berlin, 1914) for a discussion of the proper classification of this organism. 



THE BLOOD. 583 

in distinguishing these points from those of specimens taken from human 
lesions. These pure cultures produce typical lesions when injected into animals, 
thus completing the chain of evidence. 1 

(7). Yellow Fever. 

This infectious noncontagious febrile disease is caused by a specific 
organism not yet discovered. This etiologic factor is, however, blood-borne, 
as shown by the fact that injection of blood from a yellow-fever patient into a 
healthy subject causes the disease. Whatever the organism be it must be very 
minute, as the blood serum retains its infecting power after passage through 
a Berkefeld filter. The various organisms which have been held responsible 
for this disease, among them the Bacillus X of Sternberg and the Bacillus 
icteroides of Sanarelli, have all been abandoned and even the myxococcidium 
stegomyiae of the Yellow Fever Commission is at present considered as foreign 
to yellow fever. 

In 1 88 1 Finlay advanced the hypothesis that yellow fever was transmitted 
to man only through the bite of a mosquito of the Culex group, the stegomyia 
fasciata. The United States Commission, consisting of Reed, Carroll, Agra- 
monte, and Lazear, furnished the experimental proof that this hypothesis was 
valid and showed that the unknown organism of the disease required a period 
of 12 days' development in the body of the mosquito before it could be trans- 
mitted from the stegomyia as an infecting agent. A second U. S. Commission, 
consisting of Parker, Pothier, and Beyer with the help of Smith stated in 1903 
that yellow fever was due to a parasite of the sporozoan type, the myxococ- 
cidium stegomyiae, which developed in the stegomyia. This organism has 
never been found in the human body, hence its schizogony (asexual development) 
is unknown. The French Commission, Marchoux, Salimbeni, and Simond, 
as well as Schaudinn and Carroll, do not believe that this organism has any- 
thing to do with yellow fever. 2 Seidelin 3 has reported the finding of a parasite, 
a protozoon, in the blood, which he believes the causative factor. 

As this disease is beyond question blood- borne, its hematological changes 
are of some interest. Jones shows that anemia is infrequent, that fibrin 
formation is deficient, that the globucidal action of the serum is greatly increased, 
and that both cholemia and hemoglobinemia occur. The red cells show little 

1 In his later work Noguchi has cultivated the organism directly from the lesions in man, 
using a high cylindrical layer of solid media consisting of two parts of 2 per cent, slightly 
alkaline agar and one part of ascitic or hydrocele fluid, at the bottom of which has been 
placed a fragment of sterile tissue. The small pieces of tissue taken from the lesion are 
inserted deep into this media and the growth carried on as outlined above. See, also, Baeslack, 
Jour. Infect. Dis., 1913, XII, 55. Noguchi has', also, cultivated various types of spirochetae, 
which closely resemble the pallida, and has thus shown their morphological variations (Jour. 
Exper. Med., 1912, XV, 466; 1912, XVI, 194, 199, 261 and 620; Ibid., 1913, XVII, 89). See 
also, Clark and Gates, Am. Jour. Dis. Child., 1915, IX, 126; Zinsser, Hopkins and Gilbert, 
Jour. Exper. Med., 1915, XXI, 213. Zinsser and Hopkins (Ibid., 576) as, also, Kissmeyer 
(Deutsch. med. Wchnschr., 1915, XLI, 306) have shown the presence of antibodies (agglutin- 
ins) for the treponema pallidum in the blood of syphilitics. 

2 See Yellow Fever, Senate Document No. 822, Washington, 191 1. 

3 Jour. Path, and Bacterid., 1911, XV, 282 Otto, in Kolle and Wassermann's Handbuch 
der pathogen; Mikroorg., 1913, VIII, 623. See, also, Wenyon and Low, Jour. Trop. 
Med. and Hyg., 1914, XVII, 369; Ibid., 191 5, XVIII, 55; Camerer, U. S. Naval Med. Bull, 
1915, IX, 65. 



5^4 



DIAGNOSTIC METHODS. 




variation in number, Pothier never finding them below 4,280,000. The 
hemoglobin suffers considerable loss, being usually between 50 and 75 per cent. 
This loss is restored very slowly in convalescence. Albertoni draws attention to 
the lack of parallelism between the percentage of hemoglobin and the 
specific gravity of the blood, the latter falling much more than the former. 

Morphological changes in the reds 
are rare. An occasional normo- 
blast may be seen. 

The leucocytes range between 
4,660 and 20,000, the higher the 
count the more favorable the prog- 
nosis. In this leucocytosis the 
polynuclear neutrophiles are in 
higher proportion, only rarely being 
normal. Eosinophiles are few and 
myelocytes only occasional. 

(8). Rocky Mountain Spotted 
Fever (Tick Fever). 

This disease is not to be con- 
fused with typhus fever or epidemic 
cerebro-spinal meningitis, to both 
of which the term "spotted fever" 
has been occasionally applied. 

Wilson and Chowning have 
reported that the blood of man 
affected with Rocky Mountain 
spotted fever shows the presence 
of an erythrocytic parasite which 
they call Piroplasma hominis. 
These parasites are ovoid in form, 
have ameboid motility, and are 
unpigmented. Three forms of 
these intracellular ovoids were 
found: (1) a small, non-motile 
form, 1 to 2 microns in length by 1 micron in width; (2) a larger actively 
ameboid form, 3 to 5 microns in length by 1 to 1.5 microns in width, and 
showing a dark granular spot at one end; (3) a twin form, consisting of 
two pear-shaped bodies, lying with their tapered ends approaching and bear- 
ing a granular spot at each end. These bodies stain best with the polychrome 
dyes. 

Through the work of Ricketts and King it has been definitely established 
that the parasite of spotted fever finds its host in the wood tick (dermacentor 
venustus (Banks) or, as Stiles states, the dermacentor andersoni). There is 
apparently no cycle of development in the tick as an intermediate host. 
Ricketts advances much evidence against the piroplasma as the causative fac- 





o. — Schistosomum hematobium; male 



with female in 
after Loos.) 



gynecophoric groove. {Tyson 



THE BLOOD. 585 

tor, but is unable to say that such might not be the etiologic unit. After much 
research he 1 succeeded in rinding an extra- and intra-cellular pleomorphic polar 
staining bacillus, which is extremely small and is constantly present in the 
blood of infected animals as well as in the infecting tick. Whether this is 
the true etiologic factor is still unsettled, but Ricketts believed it was. 2 

The red cells in this disease are reduced to about 4,000,000, while the hemo- 
globin content may be as low as 50, thus giving a low color index. Degenerations 
and atypical staining qualities are rare. The leucocytes are increased to 12,000 or 
more and show nothing differentially abnormal, except a slight increase of the 
large lymphocytes. 

(9). Distomiasis (Bilharziasis) . 

This is a chronic parasitic disease due to the deposition in the tissues of the 
eggs of the worm, Schistosomum hematobium, also called Bilharzia hematobia, 
gynecophorus, distomum hematobium, distoma capense, and Thecosoma. It is 
a very common condition in Africa, but has been found but six times in America, 
according to O'Neil. Infection appears to be more commonly induced by drink- 
ing unfiltered infected water, but occasionally it may come through the skin. 3 

The adult parasites inhabit the blood of the portal vein and the vessels 
of the pelvis, rectum, and bladder. The male is smaller and thicker than the 
female, is 12 to 15 mm. long and 1 mm. broad, is flat and so folded as to form 
a gynecophoric canal which holds the female. The female is about 20 mm. 
long and 0.25 mm. thick and is the active agent in the infection. 

The eggs of the parasite are oval or spindle-shaped, measure about 0.16 mm. 
in length and 0.05 mm. in breadth, and have a distinct spine-like projection from 
the posterior end or from one side. These ova are particularly frequent in the 
urine of such cases. Occasionally they may be found in the circulating blood. 

VI. Bacteriology of the Blood. 

From the standpoint of differential diagnosis and treatment of a con- 
dition known or thought to be of infectious origin, it is frequently of especial 
importance carefully to examine the blood for the presence of various bacteria. 
Moreover, in many conditions, as for instance in typhoid fever, cultures made 
from the circulating blood often give positive findings long before other tests are 
obtainable. The bacteriological study of the blood requires not only a thorough 
working-knowledge of the principles underlying the bacteriological technic but, 
in many cases, special training, also, in the isolation and differentiation of the 
suspected organism. 4 
Technic. 

The success of this work depends partly upon the obtaining of a suffi- 
cient quantity of blood and partly upon the selection of the culture-media for 

1 Jour. Am. Med. Assn., 1909, LII, 379. 

3 See McClintic (Pub. Health Rep., 1912, XXVII, 732) for an excellent bibliography of 
the subject; also, Rucker, Ibid., 1912, XXVII, 1465. 

3 See Looss, in Kolle and Wassermann's Handbuch d. pathogen. Mikroorg., 1913, VIII, 1; 
Kartulis, Ibid., 23; Bovaird and Cecil (Am. Jour. Med. Sc, 1914, CXLVIII, 187) report two 
cases of infection with the closely related schistosomun japonicum. 

4 See Cummer, Ohio State Med. Jour., 1914, X, 201; Lintz, New York Med. Jour., 1915, CI, 
660. 



586 DIAGNOSTIC METHODS. 

the future development of the organisms present. The amount of blood re- 
quired varies from 5 to 20 ex., so that it must be obtained by venous puncture. 
The vein usually selected is the median basilic or the median cephalic at the 
bend of the elbow, but, if these be not available, the veins of the dorsum of the 
hand may be used, although the latter are less satisfactory. The site of puncture 
should be carefully cleansed, using all the precautions taken for any surgical 
procedure. As a rule, contaminations from the skin are rare providing this 
cleansing has been adequate. In some cases, especially in very obese patients, 
it is necessary to expose the vein by incision of the skin and subcutaneous fat 
over the vein. 

The instrument best adapted for venous puncture is, in the writer's opinion, 
the Ltier syringe, which is made entirely of glass and has a tightly fitting 
platinum needle. 1 Sterilization is best done by the use of dry heat to 150 C. for 
one hour, boiling in 1 per cent, sodium carbonate solution for 45 minutes, or 
using steam under pressure in an autoclave for 15 minutes. These instruments 
should never be sterilized with carbolic acid or bichlorid solution owing to the 
danger of inhibiting the growth of the suspected organisms. It is well, also, to 
sterilize a pair of forceps to use in fitting the needle on the syringe. The properly 
sterilized outfits should be kept in long test-tubes, plugged with cotton, the 
point of the needle being protected by resting upon a pad of sterilized cotton. 

When the above precautions have been taken, an elastic band or a towel 
may be fastened about the arm above the point of puncture, to produce con- 
striction of the vessels and distention at the point of puncture. The skin may be 
anesthetized with a spray of ethyl chlorid, if the patient be very susceptible to the 
slight pain caused by the puncture. The needle is plunged into the vein against 
the direction of the blood current, care being taken that the point of the needle 
is sharp as, otherwise, the vein may roll about beneath it. Very slight aspiration 
is necessary to draw the blood into the syringe, as the blood tends to flow 
into the instrument through its own force. The usual amount withdrawn is 10 
c.c. but occasionally 20 c.c. are preferable, especially if the patient is plethoric 
and can stand the loss easily. When the desired amount is obtained, the band- 
age is removed before the needle is withdrawn. 

After withdrawal from the vein, the needle is quickly removed from the 
syringe and the blood divided among several tubes containing agar melted and 
cooled to 40 C, the tip of the syringe being passed through the flame before 
inoculating each tube. In deciding as to the amount of blood to add to the agar, 
a general rule is to increase the amount of blood when the feeble-growing 
organisms, such as the pneumococcus and gonococcus, are suspected. The 
usual proportions are 2 or 3 of blood to 5 of agar. It is, probably, preferable 
to add the blood to fluid media, such as bouillon or litmus-milk, using 1 or 2 
c.c. of blood to 100 c.c. of media in order to overcome the normal bactericidal 
effects of the blood. 2 The blood and fluid media are then directly incubated at 

1 See Judd and Simon (Jour. Am. Med. Assn., 1915, LXTV, 822) for the use of the Keidel 
tube charged with culture medium. 

2 Hiss and Zinsser (Text-book of Bacteriology, p. 126) recommend the addition of a 
small piece of calcium carbonate to the liquid media in order to neutralize any free acid formed 
in case the pneumococcus is suspected. See, also, Gillespie (Jour. Exper. Med., 1913, XVIII, 
584) for comparative viability on fluid and solid media. 



THE BLOOD. 587 

37 C. In the case of the agar tubes, the blood and media are mixed by shaking 
as quickly as possible, poured into Petri dishes and placed in the incubator. 
It is advisable to use both solid and fluid media. Anaerobic cultures are pre- 
pared as usual, using, preferably, a combination of a vacuum with alkalinized 
pyrogallic acid solution to insure absence of oxygen. 

If, after incubation for 24 hours, the plates show only a few surface col- 
onies, contamination is to be considered, while if the colonies be deep and 
are observed in several plates, contamination may be ruled out. This must not 
be interpreted to mean that any colonies will, necessarily, develop within 24 
hours, as the plates may have to be kept for 5 to 10 days before a negative result 
may be certain. However, the contaminating organisms usually show up within 
24 hours. The fluid media will become cloudy if any organisms are present. 
From the plate colonies and the fluid media sub-cultures are then made upon 
different media and every expedient possible used to isolate and identify the 
offending organism. Usually the infecting organism may be obtained in pure 
culture as a mixed infection in the blood is very uncommon. 

Organisms Found in the Blood. 
1. Bacillus Typhosus. 

Instead of transferring the blood, withdrawn from the vein, to bouillon 
or melted agar, one may add it directly to sterilized ox-bile in the propor- 
tion of 1 or 2 c.c. of blood to 5 of the bile. The blood may, if desired, be 
obtained in this case from a puncture of the ear (the puncture being prefer- 
ably made through a coating of collodion and the blood allowed to drop directly 
into the tube of bile). The mixture of bile and blood is allowed to incubate 
at 37 C. for 12 to 15 hours, when transfers of a few loopsful are made to the 
Drigalski-Conradi or modified Endo media discussed under Feces. The char- 
acteristic colonies are then sub-cultured and the agglutination tests made as 
described in the next section. 

According to Kolle and Hetsch, 1 this bacillus of Eberth-Gaffky shows 
the following characteristics: it is an actively motile bacillus with numerous 
flagella, stains with the ordinary dyes but is Gram-negative, produces no 
indol in bouillon (colon bacillus does), produces no gas in glucose-bouillon 
(colon bacillus does), produces no acid nor any coagulation in litmus-milk 
(colon bacillus produces much acid, while the bacillus faecalis alcaligenes is a 
strong alkali-former), produces no change in neutral red-agar (colon bacillus 
and paratyphoid bacillus B cause fluorescence of the solution and gas formation, 
while the bacillus faecalis alcaligenes and the dysentery bacillus produce no 
effects), produces dew-drop colonies on litmus-lactose-agar with no change in 
color of the media (colon bacillus produces marked red coloration), produces 
acid and coagulation of the casein in litmus-nutrose-glucose solution, while no 
such effects are observed in litmus-nutrose-lactose solution, shows characteristic 
agglutination with pure culture of typhoid bacillus. 

Ordinarily the agglutination tests are all that are necessary after the 
organism has been grown on the Drigalski-Conradi or other media. However, 

1 Die Experimentelle Bakteriologie. Berlin, 191 1. 



588 DIAGNOSTIC METHODS. 

it is to be remembered that the freshly isolated organisms show less tendency to 
agglutinate than do the organisms which have been grown for some time or have 
been transferred through several generations. 

The importance of blood cultures 1 in cases of typhoid fever may be esti- 
mated by the experimental evidence that 70 to 80 per cent, of cases are positive, 
oftentimes many days before the agglutination tests are positive. It has been 
further shown that the organisms tend to diminish in the blood during the 
course of the disease until they practically disappear from the blood about the 
end of the third week. 

2. Bacillus Paratyphosus. 

In certain cases, which clinically closely resemble typhoid fever, although 
running a somewhat milder course, organisms may be isolated from the blood, 
which are intermediate between the true bacillus typhosus and the bacillus 
coli communis. The clinical differentiation of these cases is often difficult, 
although the sudden onset of vomiting, diarrhea, chill, and fever of paratyphoid 
are somewhat different from the more gradual onset and step-like increase of 
temperature in typhoid; while the temperature-curve, the somewhat different 
early characteristics of the feces, the early appearance of herpes, the usual lack 
of splenic enlargement, etc., may serve as differential points in favor of para- 
typhoid. 2 Aside from this type, we find sporadic or epidemic cases in which the 
symptomatology is that of a severe gastroenteritis, the so-called "infectious 
meat-poisoning" usually classed as ptomaine poisoning, which is due to the 
bacillus paratyphosus. These organisms are widely spread in the animal king- 
dom and may be found even as a contamination in water supplies. Not in- 
frequently it is found in man, as a pure saprophyte, no clinical symptoms trace- 
able to its presence being observed. It may be present in the pus of otitis media, 
orchitis, cholecystitis, osteochondritis, arthritis, lymphadenitis, etc., also, as a 
secondary invader, in scarlet fever, measles, pneumonia, meningitis, etc. 

The examination of the blood is made as previously described, transfers 
and sub-cultures being made upon the various media. These paratyphoid 
bacilli are, apparently, of two types, known as "A" and "B". Both types differ 
from the bacillus typhosus in that they produce gas in glucose media and show 
different agglutination reactions. Type A behaves in most other respects like 
the typhoid bacillus, except that the former causes fluorescence in neutral-red 
agar. Type B is differentiated from the typhoid bacillus and from type A of 
the paratyphoid bacillus by not coagulating litmus-milk but in producing acid 
(red coloration) and, after 5 to 10 days, in causing a conversion of this red 
color to a deep blue (due to alkali formation) . The typhoid, colon, paratyphoid 
A and dysentery bacilli never cause a blue coloration of the milk, while the 

1 This work is fast becoming an absolute necessity, as the employment of the preventive 
inoculation for typhoid practically destroys the reliability of the Widal test of the serum of 
those previously inoculated. See Moreschi, Ztschr. f. Immunitatsforch., 1914, XXI, 410; 
Mcintosh and McQueen, Jour. Hyg., 1914, XIII, 409; Saski, Ztschr. f. klin. Med., 1914, 
LXXX, 79; Carnot and Weill-Halle, Presse med., 191 5, XXIII, 89; Hohlweg, Munch, med. 
Wchnschr., 1915, LXIT, 538; Lipp, Ibid., 539. 

2 See Hunt, Arch. Int. Med., 1913, XII, 64; Heigel, Wien. klin. Wchnschr., 1915, XXA1II, 
57; Dreyer, Walker and Gibson, Lancet, 1915, I, 324 and 643; Irons and Jordan, Jour. Infect. 
Dis., 1915, XVII, 234; Robinson, Jour. Med. Research, 1915, XXXII, 399. 



THE BLOOD. 589 

blue color due to the bacillus faecalis alcaligenes is produced at once. The 
peculiarities of the agglutination reaction with this organism will be discussed 
in the next section. 

3. Bacillus Coli Communis. 

This organism is, probably, not a unitary organism, as many types of 
the species exist. The colon bacillus is found in all warm-blooded animals and 
in many cold-blooded ones as a normal habitant of the intestinal canal. These 
bacilli show a marked antagonism for the putrefactive bacteria, so that, physio- 
logically, they should be regarded as conservative organisms. Owing to the 
wide occurrence of this organism, it is frequently found as a contamination of 
food and water supplies unless special precautions are taken to exclude it. 

Although ordinarily a harmless type, yet, from the pathologic stand- 
point, the colon bacillus is generally regarded as a pathogenic organism under 
certain conditions. In this connection we must distinguish an endogenous 
from the exogenous colon infections. The first are found in cases of wound 
infection and, more especially, in infections of the lower urinary tract into which 
the bacilli have gained entrance through the urethra; while the latter are 
observed under the following conditions. So long as the natural protective and 
bactericidal agents of the body are intact and so long as the intestinal epithe- 
lium is unimpaired, the colon bacilli do not pass into the organs or serous 
cavities of the body. However, in the course of any disease, such as typhoid, 
cholera and severe non-specific enteritis, in which the epithelium is destroyed, 
the colon bacillus may pass from the intestinal tract, either by direct contiguity of 
tissue or through the blood current, and assume a pathogenic role as a secondary 
invader. Moreover, should the patient be suffering with a chronic wasting 
disease which lowers the general resistance to infection, the continuity of the 
epithelium may still be preserved and the bacillus become pathogenic in many 
organs. Thus we find infections due to the bacillus coli communis in the kidney, 
urinary bladder, gall-bladder, peritoneal cavity, ovary, uterus, and other organs. 
Pus pockets, which may have caused little clinical disturbance, may spread, 
become chronic and induce a septicemia, of which the etiologic factor may be 
determined only by resort to blood culture. 

The biologic properties distinguishing the colon bacillus from the typhoid 
and paratyphoid bacilli are as follows: The colon bacillus is very little, if 
at all, motile; it produces gas in glucose media and indol in bouillon or peptone 
media; it coagulates milk and produces acid with red coloration in litmus 
milk; its colonies on litmus-lactose agar are deep-red in color (the color diffus- 
ing into the surrounding media), while those of typhoid and paratyphoid 
are colorless; in neutral-red agar it causes fluorescence and marked gas pro- 
duction, while the typhoid bacillus produces no change in the media; it does 
not show characteristic agglutination reactions, while the typhoid bacillus does. 

4. Pneumococcus. 

This organism may be recovered from the blood in practically all cases of 
lobar pneumonia due to infection with this type, although it must be remem- 
bered that other organisms may be the etiologic factor in some cases. At 



590 DIAGNOSTIC METHODS. 

times the pneumococcus may be obtained as early as 12 hours after the initial 
chill, while in other cases it may appear as late as 48 hours after the crisis. 
Rosenow obtained positive results in 160 out of 175 cases, Prochaska 48 out of 
50, while Cole and Lenhartz report only 30 per cent, of positive findings. 1 The 
finding of the pneumococcus in the blood does not, necessarily, indicate an un- 
favorable prognosis, although a large percentage, 40 to 50, of the cases in which 
it is found result fatally. 2 The earlier the organisms are found the more grave 
the prognosis. The number of organisms found by culture is not as large as in 
other types of blood infection, so that Fraenkel is lead to believe that the 
prognosis is bad when a large number of colonies develop from 1 c.c. of blood. 
The infection may spread through the lymphatic channels to the blood, so 
that we may find these organisms in the bile, urine, milk and other body 
secretions. Moreover, we may have many infections in other organs as a com- 
plication of the original pneumonia. 

Oftentimes, we find the pneumococcus as the cause of primary patho- 
logical conditions in many organs, when there has been no antecedent pneu- 
monia. Thus we may have a pneumococcic otitis media, bronchitis, pleuritis, 
peritonitis, meningitis, endocarditis, pericarditis, orchitis, tonsillitis, cystitis, sal- 
pingitis, enteritis, conjunctivitis and keratitis. The causative factor may be dis- 
covered only by examinations of the various excretions related to the special 
condition. While the blood culture may be of little clinical value in the diagnosis 
of lobar pneumonia as the clinical findings are usually so clear-cut, yet it may 
be the only means at our disposal in solving the mystery of an obscure infection 
in other parts of the body, as for instance in a malignant endocarditis. 3 

The blood is taken as previously outlined and is transferred to agar, 
blood-agar, plain, serum or ascitic bouillon, or litmus-milk. The organisms 
which are especially confusing from the standpoint of differentiation are the 
various types of streptococci. On blood-agar plates the pneumococcus shows 
an especially characteristic greenish hemolytic colony. The streptococcus 
viridans produces the same green zone, while the streptococcus pyogenes 
shows no green color but a large colorless hemolytic zone. Blood bouillon shows 
no hemolysis with either the pneumococcus or streptococcus viridans, while a 
marked burgundy-red color appears in the presence of streptococcus pyogenes. 
The differentiation between the pneumococcus and streptococcus viridans is 
made by the use of bile acids. Bouillon cultures of pneumococcus are cleared 
while those of the viridans are unaffected. Further, the pneumococcus ferments 
inulin solutions, while most strains of streptococci do not produce this result. The 
detection of the capsule and the agglutination reactions clear up the diagnosis. 

Rosenow' s Capsule Stain. 
This method 4 is, in the writer's opinion, the best at our command for 
this purpose. The technic is as follows: Make a thin smear on a perfectly 

1 See Lyall, Jour. Am. Med. Assn., 1912, LVIII, 1841. 

2 See Hastings and Boehm, Jour. Exper. Med., 19 13, XVII, 239. 

3 See Rosenow, Illinois Med. Jour., 1912, XXI, 425; Jour. Infect. Dis., 1912, XI, 94; 
also, Cole (Jour. Am. Med. Assn., 1912, LIX, 693), for a discussion of immunity in pneu- 
monia; Cole, Arch. Int. Med., 19 14, XIV, 56. 

4 Jour. Infect. Dis., 19 11, IX, 1. 



THE BLOOD. 59 I 

clean slide or cover-glass. If the material, such as sputum, is too thick, add 
enough distilled water so that it can be spread evenly by means of a piece of 
fine tissue or cigarette paper. In cases of cultures (blood-agar, serum, glucose 
or Loeffler's blood-serum being preferable), remove a small amount of the growth 
from the surface of the medium and at once mix thoroughly with a loopful of 
serum on the slide, or, better still, make a rather dense suspension in a few drops 
of distilled water and then mix an equal quantity of this suspension with serum, 
and spread by means of tissue paper. As the smear becomes nearly dry, cover 
for 10 to 20 seconds with 5 to 10 per cent, aqueous solution of tannic acid; 
wash in water and blot; stain with carbol (saturated alcoholic solution gentian 
violet, 1 part, 5 per cent, aqueous carbolic acid, 4 parts) or anilin gentian violet 
half a minute to a minute, heat over flame but do not boil; wash in water again; 
Gram's iodin solution for 1/2 to 1 minute; decolorize in alcohol (95 per cent.); 
stain for 2 to 10 seconds, depending on the thickness of the smears, with saturated 
alcoholic (60 per cent.) solution of Griibler's eosin; wash in water and blot 
finally, clear in xylol and mount in balsam or examine directly. If the organism, 
like the Bacillus mucosus, is Gram-negative, the bacillus may be stained with 
Loeffler's or aqueous methylene blue. 

The pneumococci are stained deeply brownish-black, sharply differentiated 
from the capsule, which is stained pink. Beautiful results are also obtained 
with the streptococcus mucosus. In the thickest part of the smear the space 
occupied by the capsule may be perfectly clear; elsewhere in the smear, if 
properly made, where the conditions are suitable for absorption of eosin, 
the capsule is stained deeply pink; not rarely a clear refraction zone (often 
mistaken for the capsule in former methods) may be seen peripherally to a dis- 
tinctly stained, often large capsule. In case of sputum in which the cocci are 
embedded in a more or less tenacious mucus, the capsules, at times, are not 
rendered stainable by the above method. In that case it is well to fix and stain 
simultaneously with the 2 per cent, aqueous tannic acid, 4 parts, and saturated 
solution of gentian violet, 1 part. This modification often gives beautiful 
results. The cocci, however, decolorize easily and the tannic acid-gentian- 
violet may be followed by carbol-gentian-violet and then the usual procedure. 
Ordinary carbol-fuchsin, diluted five to ten times, and aqueous eosin (50 per 
cent, saturated solution) may also be used to stain the capsule, although the 
saturated alcoholic (60 per cent.) eosin has given the best results. Decolorization 
after the modified Gram procedure of tannic acid-fixed smears is more rapid than 
in the case of heat-fixed smears, which fact should be borne in mind. 

By the use of this method it has been determined that the capsule of the 
pneumococcus and allied organisms is not difficult to preserve or readily soluble 
in water, as is generally believed. To stain the capsule is a problem of render- 
ing it stainable rather than one of preservation. The reactions which accomp- 
lish this appear to be colloidal reactions. 

5. Streptococci. 

In general infections with any of the varieties of the streptococcus, blood 
cultures reveal the offending organism in a large percentage of cases, de- 
pending, of course, upon the location and severity of the process. There 



592 DIAGNOSTIC METHODS. 

are many varieties of streptococci, of which some are saprophytic and some 
pathogenic. Pathologically, streptococci are found in the blood in many con- 
ditions. They may obtain entrance to the blood by rupture of a streptococcic 
abscess into a vessel. Infected thrombi, as met with in puerperal sepsis, may 
form the source of the invasion. Long-standing infections, such as empyemas, 
may so lower the resistance of the patient that a septicemia may result. An 
especial point of invasion is through wounds, the infection here often being 
extremely dangerous. A further type is the cryptogenetic septico-pyemia in 
which the source of infection can not be traced. 

The organism may be obtained from the blood in practically all cases 
of mycotic endocarditis due to this streptococcus. In puerperal sepsis the 
results are not so frequent, only about one-fourth of the cases being positive. 
The cryptogenetic types associated with metastatic lesions in the joints and 
organs show a much larger percentage of positive findings. In scarlet fever we 
find, according to Hektoen, that streptococci may occasionally be found in the 
blood in cases which run a short, mild, and uncomplicated course; they occur 
with relatively greater frequency in the more severe and protracted cases in 
which there may develop local complications and signs of general infection, 
although they may not be demonstrable in fatal cases. The streptococci may 
also be found in diphtheria, measles and small-pox especially in the fatal cases, 
in which they act as secondary invaders. It is to be emphasized that the prog- 
nosis is not, necessarily, bad when streptococci are found in the blood, although 
there is a large mortality. 

The morphology of these organisms is variable and the varieties numerous. 1 
The pathogenic types, isolated directly from pathologic lesions, appear to con- 
sist of rather long chains of more than 8 pairs of cocci when grown in bouillon, 
while the non-pathogenic types form shorter chains. Involution forms are 
common in specimens from culture. Some of them are encapsulated, two 
organisms being enclosed. This may lead to confusion with the pneumococcus 
but the differential points given under the pneumococcus will serve to distinguish 
them. Usually the microscopic examination of the stained specimen will be all 
that is necessary, but occasionally careful work is needed to identify them. In 
doubtful cases, 1/2 to 1 c.c. of the blood withdrawn by venous puncture maybe 
injected intraperitoneally into a mouse, when a general streptococcic septicemia 
will ensue. 

Many other organisms have been and may be isolated from the blood. 
Thus the staphylococci are not infrequently the cause of a bacteriemia arising 
from an endocarditis, osteomyelitis, or furunculosis. The gono coccus is often 
found in cases associated with gonorrheal endocarditis, arthritis, parotitis, 
myositis, etc. The meningococcus, the influenza bacillus, bacillus mucosus, 
bacillus of anthrax and of glanders have been obtained from the blood in suitable 
cases. The tubercle bacillus has been repeatedly found in cases of both acute 

1 See Winslow, Jour. Infect. Dis., 1912, X, 285; Jungmann, Deutsch. Arch. f. klin. Med., 
1912, CVI, 283; and Bergey, Jour. Med. Research, 1912, XXVII, 67. See p. 40 for a 
discussion of streptococcic sore- throat. Also, Thalhimer and Rothschild, Jour. Exper. Med., 
1914, XIX, 429 and 444; Simons, Quart. Jour. Med., 1914, VII, 291; Rosenow, Jour. Am. Med 
Assn., 1 914, LXIII, 903; Oille, Graham and Detweiler, [bid., 191 5, LXV, n 59. 



THE BLOOD. 593 

and chronic tuberculosis. However, much recent work since the publication of 
Rosenberger, who attempted to prove that tubercle bacilli were present in the 
circulating blood of incipient tuberculosis and that a primary bacteriemia was 
always present in these cases, has absolutely refuted this finding and has em- 
phasized the importance of eliminating the possibility of the presence of acid- 
fast bacilli in the reagents and, even, in the distilled water used. 1 

It is probably true that the organism at the bottom of every infectious 
disease of known origin may be obtained from the blood at some stage of the 
infection. It is important, therefore, that the clinical examination include 
blood cultures in any case of obscure origin, as in this way treatment and prog- 
nosis will be markedly influenced. Churchill and Clark 2 have recently called 
attention to the importance of this work in children. 

VII. Serum Pathology. 

This section of hematology is very closely associated with pathology and 
biochemistry and can, therefore, be taken up only in brief outline. 3 As a matter 
of fact, changes in the number of the cells as well as in the percentage of hemo- 
globin must be dependent, to a certain extent, upon the more obscure changes 
which are taking place in the plasma in various diseases. Our ability to fathom 
the secrets of the many physical and chemical changes of the plasma has been so 
slight that we have hitherto neglected to take into consideration anything but the 
changes in the cells, which can be so easily studied by the various methods 
previously outlined. Our knowledge of the various types of immunity to in- 
fection and of the many factors concerned therein has increased to such an 
extent in recent years that a complete discussion is out of place here. By the 
elaboration of the side-chain theory of Ehrlich and of the opsonic, theory of 
Wright, we have come somewhat nearer to a proper realization of the impor- 
tance of the serum in all infections as well as in many diseases in which great 
metabolic disturbance is evident. For that matter one can hardly imagine a 
condition in which the blood plasma may not show some characteristic change, 
inasmuch as the nutrition of the entire body can come only through the blood. 
When one considers the close correlation of the various organs he may see at 
once that pathological changes in any of the viscera may result in an abnormal 
blood, which may show no variations at present capable of detection. For 
these reasons one hails with delight any advance in serum pathology and 
could but wish that his knowledge might more rapidly increase. 

1 See Berry, Jour. Infect. Dis., 1914, XIV, 162; also, Rautenberg, Berl. klin. Wchnschr, , 
1914, LI, 348 and 492; Baetge, Deutsch. med. Wchnschr., 1914, XL, 591; Faber, Jour. Am. 
Med. Assn., 1914, LXIII, 1656; Jousset, Bull. l'Acad. de Med., 1915, LXXIII, 203; Lucciarini, 
Rif. Med., 1915, XXX, 253 and 281; Austrian and Hamman, Bull. Johns Hopkins Hosp., 1915, 
XXVI, 293; Kessel, Am. Jour. Med. Sc, 1915, CL, 377; Honeij, Jour. Infect. Dis., 1915, XVII, 
376. 

2 Am. Jour. Dis. Child., 1911, I, 193. 

3 See Kraus and Levaditi, Handbuch der Technik und Methodik der Immunitats- 
forschung, Jena, 1909-1911; Citron, Die Methoden der Immunodiagnostik und Immuno- 
therapie, Leipzig, 191 2; Dieudonne, Immunitat, Schutzimpfung und Serum therapie, Leipzig, 
1913; Vaughan, Protein Split Products in Relation to Immunity and Disease, Philadelphia, 
1913; Kolmer, Infection, Immunitv and Specific Therapy, Philadelphia, 1915. 

38 



594 DIAGNOSTIC METHODS. 

Ehrlich's Side-chain Theory. 1 

The early work of Ehrlich, published in 1885, advanced a theory to account 
for various phases of immunity, especially of the action of the blood in produc- 
ing antitoxins against various poisons elaborated by infectious agents. It is 
to be said that no such formation of antitoxin against the ordinary medicinal 
poisons has been found. 

According to this theory, the protoplasm of the cell consists of a central 
group of molecules (Leistungskern), in which the inherent vital characteristics 
of the cell are located and whose integrity is necessary for normal cell life. 
At different portions of the cell certain other molecular groups are attached 
exactly as side-chain groups are attached to the benzene nucleus of organic 
chemistry. These groups or, as Ehrlich styles them, side-chains are capable 
of uniting with various material which is brought into intimate relationship 
with the cell structure. Such materials are foods, toxins, and other injurious 



s Toxin united with cell. 



Receptors. 



^&- Toxophore. 

1/ <J Haptophore. 
Toxin molecule. 



Body cell. 
Fig. 151. — Illustrating the mechanism of the toxin-cell union by the intermediation 
of receptors. (Da Costa.) 

agents. In order that food may be taken up by the cell it must possess certain 
groups which will enable it to combine with the groups in the side-chain of the 
cell. It must be, in other words, homologous or, as Ehrlich states, must bear 
the same relationship to the side-chain which Fischer has applied in his assump- 
tion of the "key in the lock" hypothesis regarding ferment action upon the 
various types of hexoses. In the nomenclature of Ehrlich the side-chains are 
styled receptors and the group of the food or of the toxin which combines with 
these receptors is known as the haptophore group. These receptors, as well 
as' haptophores, possess specific affinity, uniting with one another only when 
homologous. 

It has been found that a toxin molecule has certain injurious effects upon 
the cell; it is, therefore, necessary to ascribe this action to other, than the 
two groups above mentioned, as the union of a haptophore with a receptor 

1 In this connection see Sachs, Die Haemolysine, Wiesbaden, 1902; Kraus and Levaditi, 
Handb. d. Tech. u. Meth. d. Immunitatsforschung, Jena, 1907-1911; Adami, Principles of 
Pathology, New York, 1910; Dieudonne, Immunitat, Schutzimpfung und Serum therapie, 
Leipzig, 1913; Kolle and Wassermann, Handbuch der pathog. Mikroorg., Jena, 191 2; Kolle 
and Hetsch, Die Experimentelle Bakteriologie, Berlin, 1911. 



THE BLOOD. 595 

would form an inert substance. Ehrlich, therefore, assumes the presence in the 
toxin 1 molecule of a second group which he styles the toxophore group, which 
exerts the untoward effect upon the cell. The toxophore group in itself can 
unite with the cell only through the medium of its haptophore group. As the 
cell becomes irritated by the presence of the toxophore group, it endeavors to 
overcome this by a new formation of receptors. According to the strength of 
the irritation, many more receptors will be found than can combine with the 
toxin material present, so that many of the extra receptors pass out into the 
circulation in the form of free receptors. This is a graphic explanation of the 
fact that the cell when irritated by toxic material elaborates substances from 
its own protoplasm, which have a neutralizing effect upon the toxic substance. 
These free receptors (haptines) form the antitoxins. They combine only with 
homologous toxin material and are, therefore, specific. Welch believes that these 

j<£i5*»j- — - — „ Toxins united 

l/f^l ,'"! • with cell. 



^^ tr -<C Toxins united with 

Cy ^ V ^^-J*-* antitoxin. 

Body cell. 
Fig. 152. — Illustrating the elaboration and action of antitoxin. {Da Costa.) 

antitoxins have a second function beside that of neutralization of toxin, namely, 
an irritating one upon the bacterial invaders so that these organisms are forced 
to elaborate similar substances for their own protection. Toxins which have 
been deprived of their toxophore group are known as toxoids and can combine 
with the receptors of the cell, exerting no untoward effect upon the cell. These 
toxoids may also unite with antitoxin through the medium of their hapto- 
phore group. Occasionally toxins are incompletely combined with the anti- 
toxins; that is, the antitoxic material is not sufficient in amount completely to 
neutralize the toxin, so that such toxins may still combine with the cells and 
exert a modified poisonous effect. Such attenuated toxins are known as toxones. 
It has been found that the injection into animals of bacteria, various 
body cells, and certain secretions of some animals, as for instance, snake 
venom, gives rise to the development of specific antibodies in the blood serum 
of the animal so treated, the substances injected being named antigens. 2 Such 
blood or serum will be found to have a lytic (destructive) action upon cells 

1 See Coca, Jour. Infect. Dis., 1915, XVII, 351. 

2 See Pick (in Kolle and Wassermann's Handb. d. pathogen. Mikroorg., 1912, 1, 685) who 
shows that true antigens are always albumin-containing colloids; Also, Elliott, Jour. Infect. 
Dis., 1914, XV, 501; Salus, Bioch'em. Ztschr., 1914, LXVII, 357; Porter, Biochem. Jour., 1915, 
IX, 1. 



596 



DIAGNOSTIC METHODS. 



similar to those injected. Such sera are specific, that is, they act only upon the 
kind of cell used in the injection. The term hemolysis has been introduced to 
express the destructive action upon the erythrocytes shown by the dissolving 
out of the hemoglobin from the red cell. The stroma or discoplasm of the 
red cell is a membrane which shows peculiar relations to diffusion of various 
materials into the cell and to the passage of hemoglobin and other cellular 
material from the cell. Its chief function seems to be to prevent, as far as 
possible, any loss in hemoglobin. If this membrane becomes permeable, 
then we must assume the action of some toxic material. The term hemolysis 
has reference merely to the abnormal loss of hemoglobin and not to any disturb- 
ance beyond increased permeability of the stroma. The stroma of these cells 
remains behind and may be seen in the centrifuged specimen as the so-called 



Union of complement, ambo- 
ceptor, and cell. 



Receptors 




[ Zymophore. 

— Haptophore. 
Complement. 



U- 



- — Complementophile. 
Cytophile. 
Amboceptor. 



Erythrocyte. 
Fig. 153. — Illustrating the mechanism of hemolysis. (Da Costa.) 



shadows. Hemolysis must, therefore, be considered as a sign of protoplasmic 
death. Substances (hemolysins) bringing about such change belong, neces- 
sarily, in the class of blood poisons. Such hemolysins are increased or lowered 
temperature, various inorganic compounds, such as distilled water, ammonium 
salts, and organic compounds, such as urea, bile acids, ether, alcohol, chloroform, 
solanin, saponin, and digitalin. The saponins are among our strongest 
hemolytic substances, acting in dilutions of 1 to 100,000. Besides these we 
have various secretions, such as those of the cobra, spider, and the bees which 
are active hemolytic agents. 

It has been observed by various workers that hemolysis is prevented when 
the serum is heated to 6o°. This points to the fact that some substance is 
destroyed which is of great importance in this process. In addition, it has 
further been found that the renewing of the activity of the old heated serum 
by adding a supply of fresh isologous serum will restore the hemolytic activity. 
It is evident, therefore, that there are two factors which must be taken into 
consideration, one is a thermostable (heat-resisting) substance, while the other 
is thermolabile (destroyed by heat). To the first of these Ehrlich has given 



THE BLOOD. 



597 



the name amboceptor and to the second the name complement. The amboceptor 
has been shown to have two haptophore groups, with one of which it unites 
to the receptor of the cell and with the other to the complement. The hapto- 
phore group which unites with the cell is known as the cytophile, while the 
one uniting with the complement is termed the complementophile. For 
hemolysis, therefore, we must have the cell receptor, the amboceptor, and 
the complement. The complement has also been shown to have two groups 
analogous to those of the toxin molecule. The first is the haptophore group, 
while the second is the zymophore group, through which the destructive action 
upon the cell is manifest. 



Receptors 




Erythrocyte. 
Fig. 154. — Illustrating the Mechanism of Antihemolysis. {Da Costa.) 
A, Interference of anticomplement with complement-amboceptor union. B, inter- 
ference of antiamboceptor with amboceptor-cell union. C, antiamboceptor-amboceptor 
union. D, anti-complement-complement union. 

The amboceptor is formed within the body as the result of cellular 
hyperactivity aroused by the irritant action of the toxic material. The comple- 
ment is probably derived, for the most part, from the leucocytes, and acts very 
much as an enzyme. It can exert its toxic action only when united with the 
cell by means of the amboceptor, so that free complement has no injurious 
effect. 

It has been frequently observed that the red cells are more resistant than 
normally, while in many cases they appear less resistant to hemolysis. This 
is explained by the side-chain theory very much as it explains the formation of 
antitoxin. These antihemolysins are formed within the blood plasma after 
inoculation with hemolytic material. The hyperactivity of the cell causes 
it to throw off two types of such bodies, namely, anticomplement and anti- 
amboceptors. The former combines with the haptophore group of the comple- 
ment and the latter with the cytophilic group of the amboceptor, each of these 
combinations making it impossible for the necessary union of cell, amboceptor, 
and complement to occur. 



598 DIAGNOSTIC METHODS. 

It has been found that frequently the serum of an animal, which has been 
injected with certain bacteria or with certain body cells, shows the peculiar 
property of agglutinating or clumping such bacteria or cells when these latter 
are added to it. This condition is known as agglutination and the agents 
bringing it about are styled agglutinins. These substances are developed in 
the blood of the animal during the process of adaptation toward the presence 
of such foreign material. Agglutinins from the standpoint of the side-chain 
theory are free receptors, having a haptophore group which unites with the 
receptor of the cell or of the bacterium, and cause agglutination through the 
presence of a second group known as the zymophore or agglutiniphore group. 
Agglutination, therefore, does not require the presence of a complement. 

In some cases, intraperitoneal injection of body fluids or of solutions of 
certain proteins into animals brings about a condition which enables the blood 
serum of such animals to cause a precipitation of the protein to which the 
animal has been adapted. This fact has been taken advantage of in formulat- 
ing a medicolegal method for the detection of blood of different animals and 
will be taken up in a later section. The precipitins consist of free receptors 
combining, by means of their haptophore group, with the receptor of the cell 
and exerting their precipitating effect through the medium of their zymophore 
or precipitinophore group. 

Phagocytosis. 

According to the theory of Metchnikoff, the leucocytes are capable of 
incorporating into their substance materials which are foreign to the blood 
in which they circulate. This process is known as phagocytosis and is one 
of the greatest protective measures which the system has for its fight against 
bacterial invasion. When the blood becomes laden with bacteria, as in the 
various infectious diseases, we find a leucocytosis in practically all cases, the 
exceptions having been previously noted. This is the natural sequence if 
the system is to rid itself of these invaders. The leucocytes are drawn by 
chemical attraction (chemotaxis) toward the bacteria and attempt to swallow 
them by throwing pseudopodia about them and drawing them into the proto- 
plasm. This is successful in many cases, while in others it is not, so that the 
question of ascendency of the leucocyte or of the bacterium will depend upon 
the degree of phagocytosis. Strangely enough much variation is shown in the 
susceptibility of different organisms to phagocytosis. Thus the pneumococcus 
at times is very difficultly amenable to phagocytosis, while at others it is easily 
acted upon. The recent work of Rosenow 1 has thrown much light upon the 
mechanism of this action. 

Opsonins. 

Realizing that there was a more definite basis for phagocytosis than was 
embraced in the older conceptions, Wright 2 introduced the idea of opsonins 
to designate the presence in the blood serum of substances which render the 

1 Jour. Infect. Dis., 1906, III, 683; also, Kite and Wherry, Ibid., 1915, XVI, 109. 
2 Proc. Royal Soc, 1903, LXII, 357. 



THE BLOOD. 599 

various bacteria subject to phagocytosis. The normal blood serum contains 
such opsonic material for the various bacteria with which it may be infected, 
but this varies greatly toward the different organisms. Thus we may find 
individuals showing much more opsonin (a higher opsonic index) toward one 
organism than toward another. This explains, in a way, the well-known fact 
that different people are variably susceptible to the same disease, while the same 
individual may be strongly resistant toward infection with one organism, but 
easily a victim of another infection. 

Regarding their chemical nature very little is known. There seems to be 
a certain amount of evidence which points to the fact that these opsonins 
belong to the class of globulins, while Simon and Lamar 1 have shown that 
they are apparently associated with the euglobulin fraction. Quite as little 
is known of the structure of the opsonins, so that it is at present doubtful 
in what position to place them in the side-chain theory. According to Hektoen, 2 
they may contain a haptophoric group which unites with the bacterial or other 
receptors and also an opsonipherous group which brings about changes in 
the cell, making it capable of phagocytosis. According to Savtchenko and 
Dean, the opsonins should be regarded as amboceptors, while Greig-Smith 
looks upon the process of opsonification as the first stage of agglutination. 
All of these theories must wait for future confirmation. The opsonins are 
thermolabile and are usually destroyed by heating for 10 minutes to 6o° C. 
They occur in all classes of vertebrates and show here a peculiar characteristic, 
namely, that the serum of different animals is capable of activating various 
organisms for phagocytosis by leucocytes of animals of different species. This 
would bring the opsonins into the same field as the agglutinins, precipitins, 
and hemolysins. 

From a clinical standpoint, opsonins are frequently found to be diminished 
in certain bacterial infections. It is, therefore, conceivable that the resistance 
of the patient or, in other words, his phagocytic power might be increased by 
the addition of substances which could act as opsonic material. Such 
substances are the bacterial vaccines of Wright, suspensions in physiological 
salt solution of dead cultures of the organism to which the patient shows a 
diminished power of phagocytosis. The relation of the phagocytic power of 
the patient, as evidenced by the number of organisms which a definite number 
of leucocytes takes up under the opsonifying influence of this serum, as com- 
pared with the same condition in the case of the serum of a normal individual, 
is known as the opsonic index toward the organism investigated. The 
number of bacteria taken up by the leucocytes of the normal individual is 
taken as one. For preparation of these vaccines see last chapter. 

According to Wright, the injection of a dose of vaccine is followed by a 
decrease in the opsonic index (the negative phase), which is of variable degree 
and duration, depending upon the dose. This negative phase is followed by 
an increase in the opsonic power of the leucocytes (the positive phase), which 

1 Bull. Johns Hopkins Hosp., 1906, XVII, 27. 

2 Jour. Am. Med. Assn., 1906, XLVI, 1407; Ibid., 1907, XLVIII, 1739; Illinois Med. Jour., 
XIII, 9; also, Zinsser and Cary, Jour. Exper. Med., 1914, XIX, 345. 



600 DIAGNOSTIC METHODS. 

is associated with improvement in the condition of the patient. The various 
doses of the vaccine should be so administered that it is never given during a 
negative phase. While a low opsonic index is the rule in chronic cases, high 
indices may be observed with active systemic manifestations of acute cases. 
As a rule, it is more generally beneficial to use a vaccine prepared from the 
discharges of the patient than it is to use an already prepared vaccine of the 
same organism. The reason for this is that so much difference exists in the 
various strains of the same organism that little or no result may follow the 
administration of a stock vaccine. It is not always easy to gauge the dosage 
of a vaccine in any particular case, as the idiosyncracy of the patient plays such 
an important role. It is better practice, therefore, to start with a small dose 
and obtain no reaction than it is to give an overdose which will prove very 
harmful. Failure to observe this precaution is, in the writer's opinion, the 
reason that tuberculin so soon fell into disrepute and is now being restored to 
such great favor. 

It has been found by various workers that the administration of a proper 
dose of a vaccine is followed by certain local symptoms, such as swelling, redness, 
and tenderness at the point of injection, while certain systemic symptoms, 
such as rise in temperature, general malaise, and pains in the joints, are 
practically always evident. It has, therefore, become the practice of many 
of our workers, to gauge the results of vaccine treatment more by the reaction 
than by the determination of the opsonic indices of their patients. 

Diagnostic Use of Vaccines. 

The vaccines discussed above are used almost entirely for therapeutic pur- 
poses. While it is highly probable that any of the vaccines might give char- 
acteristic reactions in cases infected with the same organism, yet they have not 
been put to extensive diagnostic use. However, certain suspensions of organ- 
isms are, at present, widely employed in this way. 

It is evident, from the preceding discussion, that the serum of a patient 
contains bodies acting as neutralizing agents against the toxins of the infecting 
bacteria. This property varies with the individual and with the severity (dos- 
age) of the infection. Patients, who have acquired immunity against certain 
infectious diseases, either by passing through an attack of the infection or by 
use of protective vaccination (small-pox or typhoid fever), or who are in the 
process of acquiring immunity, that is, are at the time passing through an active 
infection, show a certain changed reactivity to infection with the same organism. 
In other words, such patients are not insensible to reinfection, but show a 
changed reactivity, as von Pirquet 1 has shown, especially as regards the time, 
quality and quantity of the reactions observed. This is the condition known as 
Allergy and is shown only by patients immune or becoming immune (infected) 
to the suspected organism. 2 Normal individuals do not show the reaction so 
quickly nor so intensely as do the infected or immune subjects, providing the 

1 Arch. Int. Med., 1911, VII, 259. 

2 See Gurd, Jour. Med. Research, 1914, XXXI, 205; Meigs, Jour. Infect. Dis., 1914, XV, 
541; Zinsser, Arch. Int. Med., 1915, XVI, 223. 



THE BLOOD. 6oi 

dosage is properly regulated, as the former have sufficient antibodies to over- 
come the effects of the amount of toxin, which will produce local or general 
reactions in the infected patient. 1 These are the basic principles underlying the 
diagnostic use of vaccines. 

Tuberculin Reactions. 
Method of Koch. 

This is the earliest method for the diagnostic use of tuberculin. The 
normal temperature variations of the patient are first determined for 24 hours 
previous to the injection. The preparation used is Koch's old tuberculin, 
which is a water and glycerin extract of the cultures of the tubercle bacillus, of 
such a strength that 1 c.c. is equivalent to 1 gram of tuberculin. 

The injection may be made into the deeper tissues either in the intra- 
scapular or gluteal region by means of a properly sterilized hypodermic syringe. 
The amount of tuberculin to be injected depends on the age and condition of 
the patient. As a rule 1/2 to 1 mg. is given to a robust adult, while a weak 
patient or a child receives 1/10 to 3/10 mg. These strengths are easily made 
by diluting the original solution with the proper amount of 1/2 per cent, carbolic 
acid solution. The temperature variations should then be followed, obser- 
vations being made every two hours. A rise of 1/2 C. (9/10 F.) within a few 
hours, or even as late as 36 hours, is taken as a positive result, although some 
observers require a more marked rise before admitting a positive finding. Few 
other symptoms are noticed but, if the dose has been large, marked malaise, 
chill, high fever, and local reactions may obtain. If no rise in temperature is 
observed after the first injection, a second or a third one may be given, the dose 
being doubled each time but exceeding, under no circumstances, 10 mg. It is 
possible that a single injection of 5 or 10 mg. may give more diagnostic results 
than repeated doses up to this amount, although it must be admitted that 
such large doses may react, in rare cases, positively in normal individuals. 

A positive result is very strong evidence of an active tubercular condition. 
A negative result is less reliable as some tubercular conditions, such as peritoni- 
tis or encapsulated foci elsewhere, may not react positively. 

Method of Moro. 

In this method use is made of a salve consisting of equal parts of old 
tuberculin and lanolin. A small bit of this salve is rubbed into the skin, pref- 
erably in the thoracic or abdominal region. After 24 to 48 hours small pale 
nodules, 1 to 2 mm. in diameter, are seen at the point of inoculation if the case 
be tubercular. A more severe reaction may be observed in the form of reddish 
miliary nodules. These nodules are limited to the point of application of the 
salve and disappear in 24 to 48 hours. Occasionally, some exudation may 
arise from the intense reactions, but this is very rare. This method is very 
simple and is very reliable. 2 

1 See Hektoen, Jour. Am. Med. Assn., 1912, LVIII, 1081; also, Vaughan, Am. Jour. Med. 
Sc, 1913, CXLV, 161. 

2 See Krumbhaar and Musser, Am. Jour. Med. Sc, 1914, CXLVII, 540. 



602 DIAGNOSTIC METHODS. 

Method of von Pirquet. 

This method 1 is very widely used, especially in cases of infection in children. 
The inner side of the forearm is cleansed with alcohol and ether and allowed to 
dry. Two drops of old tuberculin are placed on the skin at a distance of 6 to 
8 cm. from each other. The skin covered by these drops is then punctured 
by a large needle or gently scarified. The tuberculin is allowed to remain for 
10 to 15 minutes, when the clothing may be replaced. It is wise to make a 
third puncture or scarification between the two tuberculin points as a control 
upon the patient's reaction, no tuberculin being used here. A slight swelling 
is usually produced which becomes surrounded by a light reddish border in 
a few hours, and disappears in 24 hours. If the reaction be positive, the 
area of puncture swells within 24 to 48 hours to a papule, reddish in color and 
10 to 15 mm. in diameter. The center may be pale and be surrounded with 
small serous points. By comparison with the normal point, a reaction may be 
more clearly demonstrated. The swelling and redness begin to disappear 
within 48 hours, a slight pigmentation usually following this positive reaction. 
No general disturbance is noted as a rule. If the reaction be repeated, a marked 
positive result may be observed as an evidence of hypersensibility, the so-called 
anaphylactic reaction. This may, also, be noticed in the other methods, even 
though a much smaller dose be used than in the initial application. 

Method of Calmette. 

This method was advanced almost simultaneously by Calmette and Wolff- 
Eisner. One or two drops of a 1 to 100 dilution of old tuberculin in physiologic 
salt solution are placed in the conjunctival sac, the lids being held apart for a 
few seconds to permit of absorption of the tuberculin. Only one eye is thus 
treated, the other being used as the control. A positive reaction appears in 
6 to 24 hours and may assume one of the following types: (1) Mere reddening of 
the caruncle and the inner surface of the lower lid; (2) the bulbar conjunctiva 
may take part in the process; (3) suppurative conjunctivitis with marked 
injection of the palpebral conjunctiva; (4) hemorrhagic conjunctivitis of the 
palpebral and bulbar portions with profuse fib rino -purulent exudation. An 
associated rise in temperature may be observed in the severe types. 2 Contra- 
indications to this test are found in any inflammation of the eye or conjunctiva, 
in the previous treatment of the eye with tuberculin, and recent application of 
tuberculin elsewhere. It would not seem that this method is advisable owing 
to the great discomfort which may arise and, especially, as it is not without 
danger of severe injury to the eye. 3 

1 See Wachenheim, Am. Jour. Dis. Child., 1912, IV, 27; Austrian, Jour. Exper. Med., 
1912, XV, 149; Rosenberg, Ztschr. f. exper. Path. u. Therap., 1913, XII, 549; and Wachen- 
heim, Am. Jour. Dis. Child., 1913, V, 466; Klopstock, Ztschr. f. exper. Path. u. Therap., 
1914, XV, 13; Monrad, Ugesk. f. Laeger, 1914, LXXVI, 1419; Kiichenhoff, Deutsch. med. 
Wchnschr. 1914, XL, 229; Salvetti, Pediatria, 1915, XXIII, 35; Fishberg, Arch. Pediat., 1915, 
XXXII, 20; Am. Jour. Obs., 1915, LXXI, 361; Kraemer, Munch, med. Wchnschr., 1915, LXII, 
5 and 46; Frazer, Med. Record, 191 5, LXXXVII, 57; Palmer, Jour. Am. Med. Assn., 19 15, 
LXIV, 1312; Cattermole, Ibid., 1915, LXV, 782. 

2 See Vaughan, Jour. Am. Med. Assn., 1913, LXI, 1591. 

* Austrian (Bull. Johns Hopkins Hosp., 191 1, XXIII, 1) advocates a similar test for ty- 
phoid fever. The antigen is a " typho-protein" prepared from a mixed culture of 80 different 
typhoid strains. Positive results are obtained in 95 per cent, of cases of typhoid while it is 
negative without exception in practically all controls. Its great advantages are that it is a 
bedside test and is given in the early stages of the disease. 



THE BLOOD. 603 

Luetin Reaction. 

Noguchi 1 has recently announced "A Cutaneous Reaction in Syphilis," 
which may prove of great value in the diagnosis of this condition. His material 
is prepared as follows: Pure cultures of the treponema pallidum (see p. 582) 
are allowed to grow for periods of 6, 12, 24, and 50 days at 37 C. under anerobic 
conditions. One was cultivated in ascitic fluid containing a piece of sterile 
placenta, and the other in ascitic fluid agar also containing placenta. The 
lower portion of each solid culture was cut out and the tissue removed. These 
agar columns, containing large numbers of spirochete, were carefully ground 
in a sterile mortar. This paste was then gradually diluted by adding, little by 
little, the fluid culture, until the emulsion becomes perfectiy liquid. This mix- 
ture is heated to 6o° C. for one hour in a water-bath and 0.5 per cent, carbolic 
acid was added. When this mixture is examined under the dark-field microscope 
40 to 100 pallidas may be seen in each field. This suspension is called luetin. 2 

The skin of the upper arm is sterilized with alcoholic sublimate solution 
before the injection. The amount of luetin injected is 0.05 c.c. to 0.07 c.c. 
This injection is intradermic, that is, in the skin as superficially as possible. 
In normal cases there appears, after 24 hours, a very small erythematous area 
at and around the point of injection. No pain or itching is experienced. This 
slight reaction gradually recedes within 48 hours and leaves no induration. In 
certain cases the reaction may reach a stage of small papule formation after 24 
or 48 hours, after which time it commences to recede. 

In positive cases the following types of reaction occur: 

(^4) Papular Form. — A large, raised, reddish, indurated papule, usually 
5 to 10 mm. in diameter, makes its appearance in 24 to 48 hours. The papule 
may be surrounded by a diffuse zone of redness and show marked telangiectasis. 
The dimensions and the degree of induration slowly increase during the follow- 
ing three or four days, after which the inflammatory processes begin to recede. 
The color of the papule gradually becomes dark bluish-red. The induration 
disappears within a week, except in certain instances in which a trace of the 
reaction may persist for a longer period. This latter effect is usually met with 
among cases of secondary syphilis under regular mercurial treatment in which 
there are no manifest lesions at the time of making the skin test. Cases of 
congenital syphilis also show this reaction. 

(B) Pustular Form. — The beginning and course of this reaction resemble 
the papular form until about the fourth or fifth day, when the inflammatory 
processes commence to progress. The surface of the indurated round papule 
becomes mildly edematous, and multiple miliary vesicles occasionally form. 
At the same time a beginning central softening of the papule obtains. Within 
the next 24 hours, the papule changes into a vesicle filled at first with a semi- 
opaque serum that later becomes definitely purulent. Soon the pustule rup- 
tures. The margin of the broken pustule remains indurated, while a crust 
quickly forms, which falls off within a few days. The induration soon disap- 
pears leaving almost no scar. This reaction was found almost constantly in 

1 Jour. Exper. Med., 1911, XIV, 557; Munch, med. Wchnschr., 191 1, LVIII, 2372. 

2 Luetin may now be obtained either from Parke, Davis and Co. or Mulford Co. 



604 DIAGNOSTIC METHODS. 

tertiary types as well as in secondary or hereditary forms which had been treated 
with salvarsan. 

(C) Torpid Form. — In rare instances, the injection sites fade away to 
almost invisible points within three or four days, so that they may be passed 
over as negative reactions. Sometimes these spots suddenly light up again 
after 10 days or so and progress to small pustular formation. 

No marked constitutional symptoms have been observed after the use of 
the luetin. In most positive cases a slight rise in temperature took place lasting 
for one day. 

The conclusions of Noguchi, 1 based upon a study of 400 cases, are as 
follows: Luetin produces a cutaneous reaction in syphilitic and parasyphilitic 
patients that is most constant and severe in the tertiary and hereditary affections. 
During the primary and secondary stages, the reaction is infrequent, and when 
present is of mild degree. An exception has been found in cases in which 
energetic treatment has been or is being carried out and in which clinical signs 
of syphilis are absent. 2 Such cases may show a severe reaction, especially those 
treated with salvarsan. In certain cases of old infection in which no treatment 
has been taken and in which no symptoms have appeared for many years, and 
in the course of which miscarriages have occurred, this reaction has failed to 
appear. Despite the absence of symptoms, mothers who have young syphilitic 
children have usually given the reaction. It remains to be determined in how 
far this reaction can be used to supplement the Wassermann reaction. It 
appears probable that the Wassermann reaction is more constant in the primary 
and secondary, and the cutaneous reaction in the tertiary and latent forms of 
syphilis. 3 It further appears that the Wassermann reaction is more directly 
and immediately affected by antisyphilitic treatment than is the cutaneous 
reaction. 4 

1 See Ziegel, Arch. Int. Med., 1912, IX, 520; Miiller and Stein, Wien. klin. Wchnschr., 1913* 
XXVI, 408; Klausner, Ibid., 973; Noguchi, Presse med., 1913, XXI, 757; Wolfsohn, Jour- 
Am. Med. Assn., 1913, LX, 1855; Kaliski, New York Med. Jour., 1913, XCVIII, 24; Schmitter, 
Jour. Cutan. Dis., 1913, XXXI, 549; Brown, Am. Jour. Dis. Child., 1913, VI, 171; Foster, 
Am. Jour. Med. Sc, 1913, CXLVI, 645; Fagiuoli and Fisichella, Berl. klin.. Wchnschr., 1913, 
L, 1811; Gavini, Ri for ma Med., 1913, XXIX, 985, 1013, 1049 and 1075; McNeil, Jour. Am. 
Med. Assn., 1914, LXII, 529; Joltrain, Ann. de Med., 1914, I, 337; Fagiuoli and Fisichella, 
Berl. klin. Wchnschr., 1914, LI, 449; Kilgore, Jour. Am. Med. Assn., 1914, LXII, 1236; 
Much, Med. Klin., 1914, X, 811; Blechmann and Delort, Ann. de Med. etChir. Infant., 1914, 
XVIII, 406; Blechmann, Delort and Tulasne, Ibid., 465; Cannata, Pediatria, 1914, XXII, 481; 
Pusey and Stillians, Jour. Cutan. Dis., 1914, XXXII, 560; Noguchi, New York Med. Jour., 

1914, C, 349; Muscel, Dersca and Friedmann, Munch, med. Wchnschr., 1914, LXI, 1272; 
Clausz, Ibid., 1933; Curti, Rif. med., 1914, XXX, 1264; Boas and Stiirup, Hospitals tid., 1914, 
LVII, 1417; Trossarello, Gazz. d. osp., 1914, XXXV, 457; Wolff and Zeeman, Ibid., 811; 
Schippers, Ibid., 817; Kafka, Berl. klin. Wchnschr., 1915, LII, 15; Ross, Jour. Ment. Sc, 

1915, LXI, 244; Sherrick, Jour. Am. Med. Assn*, 1915, LXV, 404. 

2 As this luetin reaction is an allergic one, it is not surprising to find that, in the primary 
and secondary stages, there are not sufficient antibodies present to produce the reaction. 
Although emulsion of ascitic agar itself may produce an intradermic reaction similar to that of 
luetin, yet this does not militate against the test as it shows a hypersensitiveness, which is in 
itself rather characteristic of syphilis. 

8 See Robinson, Jour. Cutan. Dis., 1912, XXX, 410. Sherrick (Jour. Am. Med. Assn., 
191 5, LXV, 404) calls attention to the fact that recent or simultaneous administration of potas- 
sium iodid will cause a positive luetin reaction in non-syphilitic individuals. 

4 Irons has recently perfected a local skin reaction in cases of generalized gonorrheal 
infections, using as his vaccine a glycerine extract of autolyzed gonococci. After a few hours 
a papule is formed with a surrounding area of hyperemia, the maximum reaction being reached 
in 24 hours. Irons classifies as positive the reactions showing a total diameter of 5 mm. or 



THE BLOOD. 605 

Schick Reaction. 

By means of this reaction individual susceptibility and immunity to diph- 
theria may be readily recognized in a great majority of cases. It consists in 
the intracutaneous injection of diphtheria toxin of definite dosage and the 
observance of the irritant action at the point of injection. 

Technic. 

In the first place the minimum fatal dose of the diphtheria toxin is de- 
termined for a guinea-pig of 250 grams weight. 1 This amount is then so 
diluted with sterile physiologic salt solution that each c.c. of the dilution contains 
0.2 of the minimum fatal dose. Of this dilution 0.1 c.c. (representing one- 
fiftieth of the minimum fatal dose) is injected into the skin by means of an 
accurately graduated syringe (1 c.c. record tuberculin syringe) with steel or 
platinum-iridium needle, which should be very fine, sharp and short-pointed. 
The site of injection is the skin of the outer side of the upper portion of 
the arm. 

Reaction. 

If the injection be properly done, one observes a white blister-like punctate- 
appearing wheal, which shows distinct markings of the openings of the hair 
follicles. This elevation (due to the traumatism) persists for a short time, all 
signs of it usually disappearing within an hour. If there is sufficient antitoxin 
in the blood of the subject to neutralize the small amount of toxin, no further 
changes should be observed. This is a negative reaction and indicates that the 
person is immune to diphtheria. 

A positive reaction is noted in the gradually increasing redness and in- 
filtration of the skin, a distinct circumscribed area of redness and infiltration 
measuring about 1 to 2.5 cm. being seen within 24 or 48 hours. This redness 
persists for several days and gradually fades, leaving a brownish pigmentation 
and slight scaling. 

In some cases, especially in older children and adults, a pseudo-reaction is 
observed, which is not due to the action of the toxin but is, rather, an ana- 
phylactic response to the protein substance of the diphtheria bacillus or, as 
Kolmer and Moshage believe, to trauma of a skin which is unduly sensitive. 
Some local redness and infiltration may be noted, but this comes on more 
rapidly than does the true reaction, the redness is less sharply outlined,the local 
infiltration is more marked and the signs disappear in 2 to 4 days with little 

over; negative when 3 mm. or less (Join:. Infect. Dis., 1912, XI, 77). See, also, Fronstein, 
Med. Obozr., 1913, LXXIX, 225. 

Gay and Force (Arch. Int. Med., 1914, XIII, 471) have introduced a skin reaction for 
typhoid fever, which consists in using a "typhoidin" solution, prepared by concentrating a 
glycerin bouillon culture of typhoid bacilli, and which is applied to the abraded skin. A differ- 
ence of 2.5 mm. between the areolae of the "typhoidin" point and the control point is taken as 
evidence of a positive reaction. See, also, Gay, Am. Jour. Med. Sc, 1915, CXLIX, 157. 

1 This toxin may be obtained from the larger laboratories handling biological products or 
may be procured from the health officers in the larger cities. In this latter case a definite 
amount of a known potency toxin is supplied together with a diluent fluid. After the toxin is 
diluted it deteriorates rapidly, so that it should not be used after 12 hours, standing. The 
undiluted toxin, obtained from the supply houses should be kept cool to prevent deterioration. 
Usually the potency of the toxin is stated on the package, so that the necessary dilution may be 
directly figured. 



606 DIAGNOSTIC METHODS. 

or no pigmentation and no scaling. These false reactions are confusing, but 
should be easily differentiated by careful observation. To control the reaction, 
one may inject- into the other arm o.i c.c. of bouillon diluted i to 10 or i to 
1,000. An extreme sensibility of the skin will be indicated by slight local 
reaction in the control arm. 

Significance. 

In normal subjects a negative result indicates, in practically ioo per cent, of 
cases, the presence in the blood of diphtheria antitoxin and, hence, an im- 
munity to diphtheria. A typical positive reaction points to an absence of anti- 
toxin and a resulting susceptibility to infection. 

From the reports of the literature, it is evident that about 80 per cent, of 
new-born children, 50 to 60 per cent, of children, especially those between the 
ages of 1 and 5, and 90 per cent, of adults contain sufficient antitoxin, as shown 
by this test, to make them immune to diphtheria. In presence of diphtheria, 
the question of exposure of other children is an important one. It is evident 
that immunization by injection of antitoxin is indicated only in those giving 
positive Schick reactions. Further, by means of this test definite separation 
of the diphtheria cases from non-diphtheritic cases may be made, as patients 
with true diphtheria have little antitoxin in the blood and hence show a positive 
reaction. Likewise diphtheria carriers may be separated from actual diph- 
theritics, as the former usually have a large amount of antitoxin in the blood 
and, therefore, give negative reactions. 

There can be little question but that this test will prove invaluable in 
hospitals in selecting internes and nurses to look after the diphtheria patients. 
Moreover, the question of isolation of actual diphtheria patients becomes a much 
easier matter, with the knowledge that those showing a negative Schick reaction 
are immune to diphtheria and need not, therefore, be removed from chance of 
infection. The economic as well as the diagnostic value" of this test seems to be 
very great. 1 

VIII. Sero-Diagnosis. 

In the attempt to enlarge our diagnostic procedures advantage has been 
taken of many of the principles previously discussed. The methods evolved 
are among those most frequently employed and are reliable within certain small 
limits which will be mentioned later. They are all based upon certain peculiar 
properties possessed by the serum of patients with infectious diseases and are, 
therefore, of special importance in differential diagnosis. 

Schick, Munch, med. Wchnschr., 1913, LX, 2608; Groer and Kassowitz, Ztschr. f. 
Immunitatsf. u. exper. Therap., 1914, XXIII, 108; Kassowitz and Schick, Ztschr. f. d. ges. 
exper. Med., 1914, II, 305; Otto, Deutsch. med. Wchnschr., 1914, XL, 542; Schick, Kasso- 
witz and Busacchi Ztschr. f. d. ges. exper. Med., 1914, IV, 83; Park, Zingher and Serota, 
Arch. Pediat., 1914, XXXI, 481; Jour. Am. Med. Assn., 1914, LXHT, 859; Veeder, Am. Jour. 
Dis. Child., 1914, VIII, 154; Kohner and Moshage, Ibid., 1915, IX, 189; Weaver and Maher, 
Jour. Infect. Dis., 1915, XVI, 342; Bundesen, Jour, Am. Med. Assn., 1915, LXIV, 1203; 
Graef and Ginsberg, Ibid., 1205; Moody, Ibid., 1206; Kolmer and Moshage, Ibid., 1915. 
LXV, 144; Zingher, Ibid., 329; Moffettand Conrad, Ibid., 1010; Birnberg, St. Paul Med. Jour., 
1915, XVII, 204; Levinson and Blatt, Arch. Diag., 1915, VIII, 201; Zuckermann, New York 
Med. Jour., 1915, CII, 808. 



THE BLOOD. 



607 



A. Agglutination Reactions. 

Since the work of Gruber and Durham and of Pfeiffer and Kolle in 1896, 
the fact has been well established that the serum of an animal, which has been 
rendered immune to certain bacteria, shows the property of agglutinating or 
clumping homologous bacteria, owing to the production of immune bodies, the 
so-called agglutinins. This reaction takes place in high dilutions of the serum 




Fig. 155. — Bacillus typhosus at beginning of Widal test. (Da Costa.) 

and is practically specific for any given organism. Widal, 1 in the same year, 
applied this principle to the diagnosis of typhoid fever, showing that clumping 
and loss of motility of the typhoid bacilli occur when a suspension of the actively 
motile types is treated with an homologous immune serum at such a dilution 
that normal or non-homologous serum does not react. 

1. Gruber- Widal Test. 




Fig. 156. — A pseudo- Widal reaction. (Da Costa.) 

Cultures. 

The cultures of the typhoid bacillus must be fresh and must show many 

actively motile organisms in the hanging-drop specimens, if they are to be used 

in the microscopic method. It is advisable always to use cultures containing 

about the same number of bacilli, as the reaction is somewhat quantitative. 

1 Bull med. Paris, 1896, X, 618 and 766. 



6o8 



DIAGNOSTIC METHODS. 



Thus, if few bacilli be present, they may be clumped by a small amount of 
agglutinin both specific and non-specific, while if the number be very large, the 
specific agglutinin may not be sufficient to cause marked clumping and loss 
of motility of the organisms. Great differences toward agglutination exist in 
different strains of the typhoid bacillus, so that it is essential always to employ, 
for diagnostic purposes, strains which have passed through several generations 
on artificial media. 

A stock culture of the bacillus is kept in sealed tubes of nutrient agar in a 
cool dark place and, from this stock, fresh agar cultures are made every few 
weeks as occasion may require. From these stock agar cultures, bouillon 
tubes are inoculated and incubated for 15 to 24 hours. These latter are used 
for the agglutination tests. These bouillon cultures should be equally cloudy 
throughout and show no pellicle formation or gross clots of bacilli. It is wise 
to make up fresh bouillon cultures every other day so as to be certain of the 
maximum motility of the organisms. 1 




Fig. 157. — A positive Widal reaction. {Da Costa.) 

In the use of the macroscopic method cultures killed by heat, formalin, 
carbolic acid, thymol, etc., may be employed. Many laboratories throughout 
the country supply these cultures for general work. Ficker's " Typhus diagnos- 
tikum" is one of the widely used types. For self-evident reasons it is wise to 
have some standard strength of these cultures. Hastings advocates a mixture 
of 5 c.c. of 5 per cent, carbolic acid solution, 10 c.c. of glycerin, and 85 c.c. of 
physiologic salt solution to which are added the organisms scraped from the 
surface of two 24-hour agar-slant cultures of bacillus typhosus (the bacilli being 
rubbed into the mixture with a spatula). Bass advises a suspension of 10,000 
million dead typhoid bacilli per c.c. in 1.7 per cent. NaCl solution to which 1 
per cent, formalin is added. 

Obtaining the Blood. 

In this procedure much depends upon the custom of the worker as to the 
method followed. As this reaction is a pure serum one, it is much more logical 
to use the serum than the whole blood, if one has any choice in the matter. 

1 Riemer (Munch, med. Wchnschr., 1913, LX, 908) calls attention to the fact that typhoid 
bacilli show diminished agglutination if grown in alkaline media. See, also, Gay and Clay- 
pole, Arch. Int. Med., 1913, XII, 621; Jour. Am. Med. Assn., 1913, LX, 1141. 



THE BLOOD. 609 

The simplest and best clinical method is the use of a small capillary tube 
(made from 1/4-inch glass-tubing) with a central bulbar enlargement into which 
the blood (15 to 20 drops) is drawn by capillarity from a puncture of the ear. 
The tube is then laid flat until coagulation occurs and the serum separates. To 
hasten this process, the end of the tube, which contains no blood, may be sealed 
in the flame and the tube centrifuged. If it is desired to send the specimen to 
the laboratory, seal both ends and pack in such a way as to insure it against 
breakage. In this connection it may be well to recall that Lyons 1 has shown the 
possibility of obtaining a culture of the bacillus typhosus from the clot settling 
in such tubes. This may be done by the method previously outlined. An- 
other method of obtaining the serum is to apply a cantharides blister which 
will furnish enough serum in 6 to 12 hours for many tests. Venous puncture 
may, also, be employed, but this is rarely advisable as sufficient serum may be 
more easily obtained in the above ways. 

Some workers use the whole blood instead of the serum. If this be done, 
the leucocytometer may be employed, using physiologic salt (0.9 per cent.) 
solution as the diluent. Draw the blood from a puncture in the ear to the mark 
1 and the diluent to 10. This gives a dilution of the whole blood of 1 to 10 or 1 
to 20 of the plasma, as the plasma forms approximately 50 per cent, of the whole 
blood. This dilution may then be used directly for the later work by mixing 
with an equal volume of the typhoid suspension. 

More frequent, possibly, in the hands of workers in municipal or the larger 
clinical laboratories, is the employment of dried blood sent to them on glass 
slides, bits of paper, mica, etc. Such specimens may be easily transported 
but can not yield anything but an approximate result, as an accurate dilution is 
out of the question unless very careful weighing of the blood is carried out. A 
large drop of the dried blood is dissolved in 10 drops of water, the dilution being 
called 1 to 10. If the blood be upon paper, the dilution must be made largely 
by guess, experience teaching an approximate dilution from the color of the 
mixture. These dilutions will, of course, be doubled when the test is applied. 

Dilution of the Serum. 

The methods of diluting the whole fresh blood and the dried blood have 
already been given. As diluting pipets for the serum, one uses small (1/4 inch) 
glass tubing which has been drawn into a long capillary at one end. A rubber 
bulb fits over the larger end and provides the suction. It is good practice always 
to have a number of these pipets on hand so that they may be discarded or 
thoroughly cleaned before a second use. If the same pipet be employed for 
different cases, a contamination may arise to such an extent that erroneous re- 
sults obtain. 

A few drops of the serum, collected as above, are drawn into the capillary 
tube by suction. From this tube the serum is then dropped into the mixing 
vessels, preferably watch crystals. Two dilutions are usually made in the 
writer's laboratory, namely 1 to 25 and 1 to 50. Allow 1 drop of serum to fall 
into each of two watch crystals, the remaining serum in the capillary being 
blown into the original collecting tube. Now add, from the same pipet used in 
1 Arch. Int. Med., 1909, IV, 64. 
39 



6lO DIAGNOSTIC METHODS. 

dropping the serum in order to insure drops of the same size, to one of these 
drops of serum 24 drops of physiologic salt solution and to the other 49 drops of 
the same diluent. Mixtures of these diluted sera with an equal volume of 
typhoid suspension, as employed in the test, give final dilutions of 1 to 50 and 
1 to 100. 

Microscopic Reaction. 

The serum (preferably), whole blood, or dried blood is diluted as previously 
described. Mixtures are then made of these dilutions with an equal volume 
of typhoid suspension. By means of a platinum loop of stiff wire, a loopful of 
the diluted serum (1 to 25) is placed on the center of a cover-slip. After burning 
the serum from the loop, a loopful of the typhoid suspension is transferred to 
the same cover-glass and the two fluids thoroughly mixed by means of the loop. 
The dilution of the serum will then, evidently, be double its original dilution, or 
1 to 50. The ordinary hanging-drop preparation is then made by inverting 
the cover-slip over the hollow of the slide, which has been ringed with vaseline. 
Instead of using the hanging-drop method, some prefer to place the cover di- 
rectly upon the slide. Usually the specimen does not dry sufficiently to intro- 
duce errors, but the results are certainly not as characteristic by this procedure 
in all cases. 

The specimen is best studied with the high-power dry lens, using a strong 
light. The organisms will be seen, in specimens properly lighted, as free actively 
motile bacilli moving with their rapid darting motion through the field. In the 
course of 45 to 60 minutes, or often in much less time, the organisms will be 
seen to be collected in clumps and to be motionless, except for a slight vibratory 
movement. This is a typical positive reaction, namely marked clumping with 
complete loss of motility in one hour at a dilution of 1 to 50. Such a reaction 
is practically never due to any other disease than typhoid fever, if the culture of 
the bacillus be a pure one. 

In some cases marked agglutination with incomplete loss of motility 
obtain. In the writer's opinion this can be interpreted only as a doubtful or 
incomplete reaction. The probability is that typhoid fever is present, but one 
must admit the possibility of group agglutinins, due to the paratyphoid bacillus, 
as well as agglutinins of other infections. When dried blood is used, a slight 
tendency to agglutination may be observed, due to the presence of small parti- 
cles of fibrin, so that in such cases the loss of motility should be demanded even 
more rigorously than when serum is used, before a positive result is reported. 
It has been shown that the serum of many healthy patients or that of subjects 
suffering with diseases other than typhoid fever frequently show a reaction 
similar to that described above. This statement is true only when the question 
of dilution of the serum is taken into account. The characteristic Widal 
reaction is shown at a dilution of 1 to 50 or higher, while in other diseases, with 
the exception of paratyphoid, the reaction practically disappears (the typhoid 
bacillus being, of course, used) at a dilution of 1 to 20. The element of time is 
also of great importance. In typhoid fever, the reaction may occur instantly 
at 1 to 10 or 20, and in one-half to one hour at 1 to 50, yet a period ranging 
from one-half to one hour is necessary with other sera at a dilution of 1 to 10 or 



THE BLOOD. 6ll 

20. On the other hand, there are cases of undoubted typhoid fever that show 
no agglutination at 1 to 10 or 20, while at 1 to 50 this is marked. What occurs 
here is uncertain, some crediting the paradox to bacteriolysis of the organisms 
at the former dilutions. 

Macroscopic Reaction. 

This method is based on the fact that the agglutination of typhoid bacilli 
by a potent homologous serum is visible to the naked eye, the clumps of bacilli 
settling to the bottom of the test-tube leaving a clear supernatant fluid. 

If living cultures of bacillus typhosus are employed, the best technic is the 
following: Make up the serum dilutions as previously described, using 1 to 50 
and j to 100 dilutions. To these diluted sera, contained in small sterile test- 
tubes (11 cm. long by 8 mm. wide), is added a loopful of typhoid bacilli scraped 
from the surface of a 24-hour agar culture. The loop is rubbed against the 
inner surface of the test-tube until the bacteria have been suspended in the 
serum. The turbid suspension is then thoroughly shaken until it becomes 
uniformly cloudy, no clumps of bacteria being seen, and is placed in the incu- 
bator at 37 C. for one hour. The tubes are then removed and examined by 
strong transmitted light. The agglutinated bacteria may easily be recognized 
by their granular flocculent appearance. General clumping should obtain in 
one hour at a dilution of 1 to 50, while complete precipitation (settling) should 
follow in 24 hours, a clear supernatant fluid being left behind. This is the 
positive reaction. A negative result is shown by no clumping and little, if any, 
loss in the turbidity of the mixture. 

For the general practitioner, who desires to use the macroscopic method, 
the employment of cultures killed by heat or chemical means is recommended, 
as it obviates the necessity of an incubator, culture media, or microscope and 
permits him to have on hand material for his tests. Any of the suspensions of 
killed organisms may be used, Ficker's and Hastings' being widely employed. 
Dilute the serum as previously described, the dilutions in this case being 1 to 25 
and 1 to 50. To this diluted serum (1/2 c.c), contained in the small test-tubes, 
add an equal volume of the suspension of killed bacteria and examine as above 
mentioned. The clumping should be observed in one hour and complete 
clearing should obtain in 24 hours, if the reaction be positive. This method 
gives very reliable results and is very simple. . 

Method of Bass and Watkins. 

These workers 1 have introduced a modification of this macroscopic agglu- 
tination test which is simple, reliable, and quick. It has the advantage that it 
can be carried out at the bedside, the result being known in 2 to 3 minutes. 
Little equipment is required. The suspension consists of 10,000 million killed 
typhoid organisms per c.c. in 1.7 per cent. NaCl solution to which 1 per cent, 
formalin is added. 

Allow a full drop of blood to fall into 4 drops of water and mix thoroughly. 
Instead of this, one may make a blood-smear (using approximately 1/4 drop of 
blood) and dissolve this on the slide with 1 drop of water, the mixture being 

1 Arch. Int. Med., 1910, VI, 717. 



6l2 DIAGNOSTIC METHODS. 

stirred with a tooth-pick or similar substance. With this diluted blood (i to 4) 
mix an equal amount of the above suspension of typhoid bacilli on a glass slide. 
Tilt the slide from side to side so as to keep the mixture flowing back and forth. 
If the reaction is positive, a grayish mealy sediment appears within one minute, 
usually in less time. This sediment appears in the fluid around the edges and 
tends to collect there. If the agitation is continued, the clumps increase in size 
for 2 to 3 minutes. If the reaction does not appear in this time, it will not show 
up at all. When the reaction is negative, no agglutination occurs and the mix- 
ture remains as clear and unchanged as when placed on the slide. 

Recently Michaelis 1 has advocated a new agglutination reaction for the 
bacillus typhosus as well as other bacteria. This is based on the fact that 
bacteria are agglutinated by acids, the rate and degree being dependent upon 
the concentration of hydrogen ions present. This test promises much in the 
future as it depends entirely upon chemical factors which are more easily and 
more exactly controlled than are the uncertain biologic units with which we 
usually work. 2 

Time of Appearance and Disappearance of the Reaction. 

The time at which a Widal reaction may appear in the course of a typhoid 
is of some importance. The earliest date of appearance of a positive reaction 
is rather hard to determine, as workers differ in their interpretation of the 
beginning of the disease. Definite reports of positive results as early as the 
second day have been given by Fraenkel and others. Although this reaction 
may appear before the rose spots, splenic tumor, or diazo reaction, yet it is 
practically never found when the blood shows a normal or an increased number 
of leucocytes, a leucopenia with a relative lymphocytosis being associated with 
a positive Widal in practically all cases. A differential blood count may, 
therefore, be of great value when the means of making an agglutination test are 
not at hand. Wood was able to obtain positive results in only 10 per cent, of 
his cases in the first week of the disease. The majority of observers are agreed 
that the first appearance of this reaction is in the second week of the disease in 
most cases. In a certain number, it may not appear till the third or fourth 
week the highest agglutinations frequently being seen toward the end of the 
disease and in the early stages of convalescence. It is very rare not to obtain a 
positive reaction at some stage of the disease, providing the tests are made at dif- 
ferent intervals. It is possible that cases reported, in which no Widal reaction 
was observed throughout the course of the disease, may be due to faulty technic 
or to insufficient tests. A single negative test, of course, proves nothing. 

The persistence of the Widal reaction is likewise variable. Little seems to 
depend upon the severity of the attack as to the time of disappearance of the 
reaction. Usually this reaction persists for 1 to 2 months, although it 
may obtain for four to 6 months or even for years. It is undetermined 
whether these latter cases are due to reinfection rather than a continuation of 
the agglutination powers of the serum. Reports are extant of cholelithiasis in 

1 Folia Serologica, 1911, VII, 1010; Deutsch. med. Wchnschr., 1915, XLI, 241. 

2 See Beniasch, Zeitsch. f. Immun.-Forsch., 1912, XII, 268; Gillespie, Jour. Exper. Med., 
1914, XIX, 28. 



THE BLOOD. 613 

which typhoid bacilli were isolated from the gall-bladder as long as 18 
years after an attack of typhoid, the Widal reaction being positive in these cases. 
In this connection it is to be said that "typhoid-carriers" do not show a positive 
Widal as a rule. 

Specificity of the Reaction. 

From the standpoint of the biologic theory, the typhoid bacillus is agglutin- 
ated only by an homologous immune serum. Practically, we find that this is 
the case within certain limits, if all conditions are properly met. A positive 
Widal reaction is not infrequently met with in cases of jaundice, in which no 
specific disease is clinically evident. * However, it is certain that most, if not all, 
of these cases of jaundice are actually secondary to a typhoid cholecystitis with its 
accompanying sequelae. Such patients may have an "ambulatory" typhoid or 
may have no clinical signs other than a positive Widal. 

Further, in many cases of infection with bacteria of the colon group, a 
"collateral agglutination" may obtain, that is agglutination of several members 
of the group may occur with one serum. Cases are undoubtedly met with in 
which the infection is due to the paratyphoid bacillus and in which the agglutina- 
tion of the typhoid bacillus is stronger than the paratyphoid; while, on the other 
hand, cases of true typhoid may show a stronger reaction with bacillus para- 
typhosus than with the bacillus typhosus. According to Lentz, the time of 
such associated agglutinations is important in this connection. The para- 
typhoid bacillus is agglutinated more quickly by a paratyphoid serum than is 
the typhoid bacillus by the typhoid serum. 

A positive reaction, all conditions outlined above obtaining, indicates 
infection with the bacillus typhosus in practically all cases. This test can not 
be regarded as of great value in the early stages, as only a very small percentage 
of cases show early agglutination reactions. It is especially serviceable in the 
differential diagnosis- from the second week of the disease on to convalescence. 
In the ambulatory types or in localized typhoid infections, especially in the 
gall-bladder, this test is of great value. A negative reaction should not be 
interpreted as excluding typhoid, as many typical and severe cases show no 
reaction until late in the disease. 2 Like all laboratory tests, it must be used only 
in connection with the clinical findings, if the best results are to follow. The 
attending physician is the one to interpret the result, not the laboratory worker. 

2. Diseases Other than Typhoid Fever. 

Other organisms, beside the bacillus typhosus, show such agglutination re- 
actions. The technic is the same and the results comparable, the same specific- 
ity being shown. It is especially in the realm of infections with members of the 
colon group that these tests are, frequently, uncertain, yet, here also, careful varia- 

1 See Mazza, Gazz. d. osp., 1914, XXXV, 921; Perlmann, Munch, med. Wchnschr., 1915, 
LXII, 435; Stuber, Ibid., 1173; Bernard and Paraf, Presse med., 1915, XXIII, 233- 

2 The widespread use of typhoid vaccine as a preventive inoculation has introduced a 
large possible error into this reaction. The serum of injected (immune) patients shows a 
positive Widal reaction very quickly. This condition persists for a varying period, so that 
blood cultures or Austrian's ophthalmo-reaction (see p. 602) should be used in such cases. 
See Moon, Jour. Am. Med. Assn., 1913, IX, 1764; also Kellermann, Miinch. med. Wchn- 
schr., 1914, LXI, 2453. 



614 DIAGNOSTIC METHODS. 

tion in the dilution of the serum and observation of the time and degree of ag- 
glutination will lead to a positive result, which will be of the greatest clinical value. 
The organisms, frequently identified by these reactions, are the paratyphoid 
bacilli, cholera spirillum, Shiga bacillus, bacillus of plague, malta-fever, tuber- 
culosis, and the diplococcus of pneumonia. l 

B. Precipitin Reaction. 

This reaction is based upon another fact well established in serum path- 
ology. It has been proven that the serum of an animal, injected with blood or 
blood-serum of another animal, shows the property, when added to an homolog- 
ous serum, of precipitating the albumin of this serum in the form of a light 
flocculent precipitate. This same peculiarity has been observed after injection 
of exudates or transudates or of pure proteins both animal and vegetable. 2 
These reactions are, therefore, specific for the type of protein injected, within 
certain definite limits. Although this test has been used in the detection of 
certain albuminous substances excreted in the urine (see p. 262), yet its chief 
interest to the laboratory worker is its application to the medico-legal detection 
of blood and blood-stains. 3 

The work of Wassermann and Uhlenhuth has shown that blood-stains may 
reveal their origin from any particular species of animal by application of this 
test. It is evident that one must, of course, prove that such stains are really 
due to blood by other methods, as this biologic test is merely for specific pro- 
teins and not for blood as such. These latter tests will be given in a later sec- 
tion. In this discussion the writer quotes largely from Uhlenhuth and Weidanz. 4 

The Anti-serum. 

This is the serum of the animal immunized against the proteins of the 
blood, exudates or secretions of a particular species of animal. These proteins 
are usually those of the human as this type is of especial importance in medico- 
legal investigations. However, in the larger laboratories where many different 
serological tests are being frequently made, animals are kept on hand, which 
have been immunized against the proteins of most domestic animals, so that an 
absolute differentiation and identification of a stain may be made. 

The preparation of the anti-serum is as follows: A rabbit is injected intra- 
peritoneally with 5 to 10 c.c. of whole blood or serum. At intervals of 3 to 
5 days other similar injections are given until six or eight in all have been 
made. About a week after the last injection, a few drops of blood are drawn 
from a puncture in the ear of the animal and are allowed to clot in a small test- 
tube. The potency of the serum is then tested by permitting a few drops of the 
separated serum to fall into a small test-tube containing about 1 c.c. of a 1 to 

1 Kolmer (Jour. Exper. Med., 1913, XVIII, 18) has shown that there is no appreciable 
amount of agglutinin for treponema pallidum in the sera of secondary and tertiary syphilis. 
See, however, Zinsser and Hopkins (Jour. Exper. Med., 1915, XXI, 576, and Kissmeyer, 
Deutsch. med. Wchnschr., 1915, XLI, 306. 

^ 2 See last chapter for a discussion of the phenomena of anaphylaxis brought about by such 
injections. 

3 See Hektoen, Jour. Infect. Dis., 1914, XIV, 403; Bing and Trier, Ugesk. f. Laeger, 1914, 
LXXVI, 2 117. 

4 Prakt, Anleitung z. Ausfuhrung des biol. Eiweissdifferenzierungsverfahrens, Jena, 
1909. See, also, Leers, Die forensische Blutuntersuchung, Berlin, 1910. 



THE BLOOD. 615 

1,000 dilution of dried homologous blood in physiological salt solution. In this 
preliminary potency test as in the later precipitin test it is important that the 
dilution of the solution under investigation should be about 1 to 1,000. One 
may recognize this proper dilution by the fact that the solution forms a layer of 
foam on shaking and, further, on heating and adding to 1 c.c. of the solution a 
drop of 25 per cent, nitric acid, only a slight opalescence obtains. If the addi- 
tion of the anti-serum to the 1 to 1,000 dilution of dried blood causes a turbidity 
in 1 to 2 minutes, the serum is sufficiently active for the test and the animal may 
be bled. 

The blood is now withdrawn, either in small amount as desired for im- 
mediate use or the animal may be completely bled. The blood is collected in 
wide-mouth test-tubes, which should be then plugged with cotton and placed 
in a slanting position until the blood is coagulated. The serum, which must be 
clear and sterile, is removed to small test-tubes, which are plugged with cotton, 
sealed with paraffin, and stored in a cool place. This is the anti-serum. It 
should be sufficiently potent to. produce a turbidity almost at once, at latest in 
1 to 2 minutes, in a 1 to 1,000 dilution of homologous blood in 0.9 per cent. 
NaCl solution. 1 

Solution of the Blood or Stain. 

As a preliminary precaution, it is to be emphasized that all vessels and 
instruments used should be thoroughly clean and sterile and that all fluids be 
absolutely clear. 

If the blood or exudate be dried upon solid material, such as glass, knife 
blades, stone, wood, etc., it is carefully scratched of! with a sterile instrument. 
This material is powdered and dissolved, as far as possible, in chemically pure 
physiologic salt solution contained in a sterile test-tube. If only traces of the 
stain are present, one may form a well of wax about the spots and place in this 
some of the salt solution. It is absolutely essential that no other solvent than 
0.9 per cent. NaCl solution be used, as otherwise erroneous reactions may occur 
or^the usual precipitin reaction may not obtain. 

If the stain has penetrated clothing or other soft material, the spot is cut 
out with scissors, is finely divided, teased with a needle and placed in a small 
watch-glass or test-tube with a small amount of salt solution for one hour, or, 
if the stain be very old or has been exposed to marked changes in the weather, 
for 24 hours in a cool place. 

The fluid obtained in either of the above ways is filtered, first through 
hardened filter-paper (Schleicher and Schiill, Nr. 575, 603, or 605) and then 
through a small Berkefeld filter. This filtrate, which must be perfectly clear, 
should be neutralized to litmus either by the addition of sodium carbonate or 
tartaric acid solutions, as the case may be. Further, this filtrate should show 
distinct foam-formation on shaking. This is not always the case if the stain 
has been exposed to the sun and dust or has dried either upon fatty material or 

1 It is important to remember that such serum may contain a small amount of antigen 
(along with the precipitin) which may persist as long as 15 days after the last injection. 
See von Dungern, Centralbl. f. Bakteriol. ite Abt., Orig., 1903, XXXIV, 355; Uhlenhuth and 
Weidanz, Loc. cit, 221; Zinsser and Young, Jour. Exper., Med., 1913, XVII, 396. 



6l6 DIAGNOSTIC METHODS. 

iron. Under these conditions, the precipitin reaction may not be seen in the 
usual time, but will obtain, as a rule, in five minutes. 

As previously stated, the dilution of this physiologic salt extract of the stain 
should be approximately i to 1,000. If sufficient stain can be scratched from 
the surface of the material upon which it is found, this may be weighed and 
proper dilution made. Such procedures are usually impossible, so that we use 
the approximate method given on previous page. 

Technic of the Test. 

The reaction is carried out in the small test-tubes, which should be ap- 
proximately of the same size and thickness, should be thoroughly clean and 
sterile, should be numbered and arranged in a series of 10 tubes in a small rack. 

In tubes i and 2 are placed 1 c.c. of the above solution of the stain to be 
tested. 

In tube 3, 1 c.c. of a 1 to 1,000 dilution of known fresh human blood in 
physiologic salt solution. 

In tube 4, 1 c.c. of a 1 to 1,000 dilution of dried human blood in 0.9 per 
cent, salt solution. 

In tube 5, 1 c.c. of sterile physiologic salt solution. 

In tubes 6, 7, 8, and 9, 1 c.c. each of a 1 to 1,000 dilution of fresh or dried 
blood of such domestic animals as may be easily obtained, as chicken, dog, 
horse and sheep blood. 

In tube 10, 1 c.c. of a physiologic salt extract of the material upon which 
the stain was found. 

To each of the above tubes, with exception of tube 2, is now added from a 
carefully graduated pipet 0.1 c.c. of the anti-serum, which has been tested for 
its potency as above described. To tube 2 is added 0.1 c.c. of normal clear 
rabbit serum. The anti-serum must be carefully added in such a way that it 
runs down the side of the tube and does not drop directly into the fluid. After 
this addition the tube must not be shaken. The reaction is carried out at room 
temperature. 

If the reaction be positive, a distinct clouding or turbidity will be observed 
immediately, or at latest within two minutes, in tubes 1,3, and 4 (all of which 
contain human blood), while the other tubes should remain perfectly clear. 
If the anti-serum has been properly added, the turbidity may be visible as a 
distinct contact ring which gradually increases upward. A negative reaction is 
shown by the lack of turbidity in any of the tubes. 

Specificity of the Reaction. 

Although this test is extremely delicate, yet it may happen that a stain, 
definitely identified as a blood-stain by other tests, may not react typically to the 
precipitin test, owing to the small amount of protein matter in the salt extract. 
This is especially the case if the protein has been rendered insoluble by heat or 
destroyed by chemical agents or by putrefaction. Even in such cases a positive 
reaction may occasionally be obtained, Uhlenhuth and Beumer reporting 
positive results after blood had been subjected to putrefaction for two years. 

As far as the influence of time upon this reaction is concerned, it may be 



THE BLOOD. 617 

said that blood-stains over 50 years old have been detected. The whole point, 
in this connection, is whether any trace of soluble protein remains. If so, the 
test will react positively, providing the dilution and the time of reaction be 
carefully regulated to suit the lessened concentration. 

Heterologous precipitin reactions do not occur if the conditions of the test 
are properly maintained. If more concentrated blood solutions are used and 
the amount of anti-serum increased, such erroneous reactions may obtain. It 
is true that confusing reactions may arise in the differentiation of the blood of the 
closely related animals such, for instance, as the horse and donkey; goat, sheep 
and ox; dog and fox; hare and rabbit; chicken and pigeon; and especially man 
and the anthropoid apes (orang-outang, gorilla and chimpanzee). However, 
the point of great medico-legal importance is that such close differentiations are 
rarely called for, as the question at issue is, usually, whether a given stain is or 
is not due to human blood. As the apes are relatively scarce in this country, 
confusion is seldom probable. This reaction may, therefore, be regarded as 
practically specific for the especial type of blood protein with which the immune 
serum reacts (all details of the test being properly regulated), only the proteins 
of the very closely related animals responding to the test in a way which might 
leave the slightest room for doubt as to the origin of a stain or of a protein. 

The substance on which the stain is found may, often, exert some retarding 
action on the test. Stains upon cloth or paper usually show prompt results, 
although those upon wall-paper may, at times, react atypically owing to the 
influence of the chemical of the dyes. Strong alkalies or acids prevent the 
reaction, but these are thrown out by neutralization before the test is applied. 
Tannic acid, derived from stains upon leather may prevent the reaction, al- 
though this is not always the case. Iron rust may retard but does not prevent 
the reaction. If the stain be upon soft clay, mortar, lime or fresh plaster so 
that an intimate mixture has occurred, the reaction will probably not obtain, 
according to Graham-Smith and Sanger, but if the stain be upon hard well- 
dried mortar no influence is seen, as demonstrated by Biondi. 

As previously stated, this active immune serum reacts promptly with the pro- 
tein elements of exudates, sputum, pus, urine, feces, nasal and bronchial secre- 
tions, vaginal, lochial and seminal fluids, etc., so that a positive precipitin reaction 
means merely that a protein is present, which has the same origin as that against 
which the animal furnishing the anti-serum was immunized. 1 Whether or not 
this is a blood protein is, in no way, a part of the test. This must be determined 
by other tests. 

C. Complement Fixation Test. 

As previously stated, the serum of an animal, which has been injected with 
bacteria or with washed blood corpuscles of an animal of a different species, 
acquires the property of dissolving homologous cells through the development 
of antibodies or immune bodies, which are specific for the cells injected. This 

1 Robertson and his pupils have contributed some interesting articles on the blood relation- 
ship of animals as displayed in the composition of the serum proteins. See Robertson, Jour. 
Biol. Chem., 1912, XIII, 325; Woolsey, Ibid., 1913, XIV, 433; Thompson, Ibid. ,^1915, XX, 
1; Briggs, Ibid., 7. 



6l8 DIAGNOSTIC METHODS. 

process is known as bacteriolysis or hemolysis. Such sera are known as lytic; 
bacteriolytic, if the action is directed toward bacteria, hemolytic, if toward blood 
cells. It is to be remembered, in this connection, that fresh blood serum of 
many species is hemolytic for the red cells of some, but not all, other species, the 
degree being relatively small. Moreover, we find that the blood of the same 
species may be markedly hemolytic for the homologous cells, as evidenced by 
the effects occasionally observed after transfusion. It is, therefore, evident that 
we may have in any serum both the immune and the natural hemolytic agents, 
the former being much the more effective. 

It has been shown that hemolysis, by means of an immune serum, may be 
prevented by heating the serum to 55 or 56 C. for one-half hour, the process 
being known as inactivation. Addition of a small amount of a fresh, isologous 
serum will restore (reactivate) the hemolytic activity. Hence, two separate 
factors must be concerned in hemolysis, the one a thermostable (heat-resisting) 
substance, the other a thermolabile (destroyed by heat) product. The first 
of these is called the amboceptor, the latter, the complement. Amboceptor is 
usually still active in serum which has been kept for a long period, while com- 
plement disappears from the serum in a short time even though it be kept on 
ice. The function of the amboceptor is to sensitize the cells, while that of the 
complement is to dissolve the cells so sensitized. Hemolysis is impossible 
without the combined action of both factors, either one singly having no effect. 
Complement can combine with the cell only through the medium of amboceptor, 
while this union does not take place unless amboceptor and antigen (the blood 
cells) are homologous. In other words, the cells can be hemolyzed only by 
homologous serum in the presence of complement. It is evident, on the other 
hand, that hemolysis must occur when the proper hemolytic trinity is present. 
The amboceptor and complement bear definite quantitative relations to one 
another in this process, an increase of one factor permitting the use of a less 
amount of the other. 

Complement may combine itself equally well with bacteriolytic or hemo- 
lytic amboceptor prodding the homologous antigen be present. If, therefore, 
we mix bacterial emulsions with the corresponding inactivated immune serum 
and complement, this latter must attach itself (become fixed) to the bacterial 
cell through the medium of the amboceptor, so that the later addition of hemo- 
lytic amboceptor and washed cells will not result in hemolysis, owing to the fact 
that the complement originally present has been fixed to such an extent that 
none is left to combine with the hemolytic amboceptor. This phenomenon, 
discovered by Bordet and Gengou, is known as " fixation of complement" or as 
"deviation of complement." It is decidedly a quantitative as well as a qualita- 
tive reaction. Had complement remained free or been added in excess of the 
amount necessary to act upon the bacterial cell, a certain amount of visible 
hemolysis must have occurred, and might, even, have been complete, had 
sufficient hemolytic amboceptor been present. These quantitative elements 
are the most important ones in the application of the test to be discussed later. 

This test has been used for diagnostic purposes in many infectious diseases; 
for instance, in typhoid fever by Widal and Lesourd as well as by Hirschfeld, in 



THE BLOOD. 619 

cerebrospinal meningitis by Cohen, in whooping cough by Bordet and Gengou, 
in scarlet fever by Besredka and Dopter and Foix and Mallein, in systemic 
gonococcic infections by Muller and Oppenheim, Bruck, Albarran and Jungano. 
Its most important and frequent application is in the sero-diagnosis of syphilis, 
as advanced by Wassermann, Neisser and Bruck and, independently, by 
Detre. 

It is evident that the application of this complement fixation test leads, 
just as in the case of the agglutination and precipitin tests, to the identification 
of an antibody, provided one has the homologous antigen, or vice versa, the 
antigen and antibody necessarily being homologous and, hence, specific, the 
one for the other. It had been shown by Klebs, Metchnikoff and Roux that 
syphilis could be transmitted to monkeys by inoculation with human syphilitic 
material (antigen). Wassermann and his coworkers then found that the serum 
of these syphilitic apes contained an antibody not found in normal serum. 
Having this antibody, it was an easy step to show its relation to syphilitic antigen 
and to apply the complement fixation test in the detection of antibodies in the 
serum of patients infected with syphilis. 1 As the cultivation of the treponema 
pallidum has only recently been achieved by Noguchi, no direct method of 
detecting these antibodies is available. 2 As indirect methods must be used,, the 
antigen employed in the early studies was an extract of syphilitic tissues in the 
active stage of the disease, as these contain large numbers of the causative 
spirochete. Later workers have shown that this antigen need not be specific, 
as alcoholic extracts of certain normal organs, crude tissue lecithin, or, even, 
salts such as sodium glycocholate or oleate, may serve quite as well in the reac- 
tion. Although this fact lessens the true specificity of the test, from the biologic 
standpoint, there is no doubt of the clinical value of the reaction. The so-called 
syphilitic antigen must be either a normal substance or one possibly associated 
with an unknown product which actually produces a specific result. However 
this may be, it is a well established fact that, if the serum of a patient infected 
with syphilis be treated with antigen and complement, no hemolysis occurs on 
the later addition of hemolytic amboceptor and red cells because the complement 
is bound to the antigen through the medium of the homologous amboceptor; 
while if the patient is non-syphilitic marked hemolysis occurs as complement 
is free to act with the hemolytic system. This is the basis of the Complement 
Fixation Test as applied to syphilis by Wassermann and his colleagues. Al- 
though the antigen need not be specific, yet the test itself is remarkably specific 
being given by only a few non-syphilitic conditions as will be seen later. ■ 

In the actual technic of the test, many modifications have been advised, 
the variations consisting either in the antigen or in the cells to be hemolyzed or 
in both. Five essential factors enter into the reaction whatever be the modifica- 
tion employed. These are (1) syphilitic antigen, (2) syphilitic antibody, (3) 
erythrocytes (antigen), (4) hemolytic amboceptor and (5) complement. These 
factors must be accurately adjusted to one another, as serious errors may arise 
if the quantitative relations are not observed. The writer will describe only 

1 See Krauss, Biochem. Ztschr., 1914, LXIY, 222. 
2 See foot-note on page 622. 



620 DIAGNOSTIC METHODS. 

the original Wassermann and the Noguchi methods, as these are, in his opinion, 
the most reliable ones to follow. 1 

a. The Wassermann Reaction. 
Preparation of Antigen. 

Since the discovery of the fact that the antigen used in this test was not 
absolutely specific, in the biologic sense, for the syphilitic antibodies, many 
different antigens have been advocated. The writer selects those which have 
promised the best results, although he believes that Noguchi's antigen and, 
especially, Sachs' alcoholic heart antigen with cholesterin are by far the most 
reliable and constant ones introduced. 

a. Aqueous Extracts. 

i. Wassermann's Method. 2 
The liver or spleen of a syphilitic fetus is ground up in a mortar with sand 
or, preferably, minced in a meat grinder. The hash is placed in a dark-colored 
flask and mixed with 0.9 per cent, sodium chlorid solution containing 0.5 per 
cent, phenol. The proportion is 1 gram of substance to 4 c.c. of the salt solu- 
tion. This mixture is shaken for 24 hours in a shaking machine and is then 
allowed to settle or is centrifuged. The reddish-brown opalescent supernatant 
liquid, which is the antigen, is decanted into dark bottles, tightly stoppered and 
kept on ice. On standing, a precipitate forms, which must not be disturbed by 
shaking. This aqueous antigen appears to be rather unstable and shows, sooner 
or later, some inhibition (anticomplementary action) with normal serum, 
although Citron 3 finds such antigens active even after 11 months. Before use 
the activity of this extract, as well as of all others, must be quantitatively 
determined by the method later outlined. The usual doses are 0.2 (maximum) 
and 0.1 (minimum) c.c. As a general rule, we use only such an amount of 
antigen, the double dose of which shows no binding of complement. 

b. Alcoholic Extracts. 

1. Method of Porges and Meier. 4 

The liver of a syphilitic or normal fetus is hashed and extracted for 24 
hours with five times its weight of absolute alcohol. Filter through filter- 
paper and distill the alcohol from the filtrate in vacuum at 40 C. A syrupy 
residue remains which is suspended in physiologic salt solution containing 0.5 
per cent, phenol, using 100 parts of salt solution to 1 of syrup. Filter through 
filter-paper. The usual dose of this antigen is 0.2 to 0.3 c.c, although less 
quantities may be used as determined by methods of standardization. 

Instead of this antigen, these workers use, also, a 1 per cent, solution of 
commercial lecithin in phenol-containing physiologic salt solution. This 
solution is stable if kept in the ice box. It has the disadvantage that the smaller 

1 Hecht (Wien. klin. Wchnschr., 1909, XXII, 265) and later Weinberg (Ann. de l'lnst. 
Pasteur, 1912, XXVI, 427) utilize the natural amboceptor and complement present in unheated 
human serum. See, also, Gradwohl, Jour. Am. Med. Assn., 1914, LXIII, 240. 

2 Deutsch. med. Wchnschr., 1906, XXXII, 745. 

3 Die Technik der Bordet-Gengouschen Komplementbindungsmethode. in Handbuch of 
Kraus and Levaditi, Jena, 1909, p. 1076; Die Methoden der Immunodiagnostik una Im- 
munotherapie, Leipzig, 1910. See, also, Bruck, Die Serodiagnose der Syphilis, Berlin, 1909. 

4 Berl. klin. Wchnschr., 1908, XLV/731. 



THE BLOOD. 62 1 

doses may inhibit hemolysis, while the larger ones may, themselves, cause 
hemolysis. The stronger luetic sera require a larger dose (0.1 c.c), while the 
weaker sera use less (0.01 c.c.) of lecithin. Hence we employ an average dose 
of 0.05 c.c, which may be compensated by the use of four to six times the 
titrated dose of amboceptor and complement. 

2. Method of Lands teiner, Miiller and Poetzl. 1 

The muscular tissue, free from fat, of a guinea-pig heart is rubbed up in a 
mortar and extracted with 95 per cent, alcohol, using 50 c.c. for every gram of 
substance. Heat for several hours at 6o° C. and filter through filter-paper. 
The filtrate is stable at room temperature for long periods. It is used in doses 
of 0.3 to 0.05 c.c. 

3. Method of Sachs. 2 

As it had been shown 3 that the addition of cholesterin to crude alcoholic 
extracts furnished an antigen of especially powerful and specific properties, 
although cholesterin itself had no antigenic effect, Sachs advocated a choles- 
terin-fortified extract of beef heart or liver or of guinea-pig heart. Mcintosh 
and Fildes 4 find that human heart furnishes a superior antigen. This latter 
material is used in the writer's laboratory, the antigen being prepared as follows: 

The muscular tissue of a human heart, normal or pathologic, is freed from 
gross fat, finely minced, weighed, and ground up in a mortar with absolute 
alcohol in the proportion of 1 gram of tissue to 10 c.c. of absolute alcohol. 
Transfer the mixture to a stoppered bottle and shake for one and one-half 
hours at room temperature. Filter through paper and preserve in an amber 
stoppered bottle*. Although the activity of this extract does not change for 
2 or 3 months, a more reliable preparation may be had by preparing it 
fresh each month. To 5 parts of this 10 per cent, alcoholic extract of human 
heart add 4 parts of a 1 per cent, solution of cholesterin in absolute alcohol. 
This forms the stock antigen. When used in the test a fresh 1 to 10 emulsion 
in normal saline is prepared by adding the stock antigen drop by drop with 
constant stirring to normal saline in proportion of 1 part of former to 9 of latter. 
Walker and Swift 5 believe that syphilitic patients under treatment should be 
treated until negative reactions obtain with a 1 to 6 dilution of this extract, as 
this gives a much more delicate reaction. The point to be remembered is that 
not more than one-fourth of the anti-complementary dose of this antigen should 

1 Wien, klin. Wchnschr., 1907, XX, 1565; 1908, XXI, 282. 

2 Berl. klin. Wchnschr., 1911, XLVIII, 2066. 

3 See Browning, Cruickshank and McKenzie, Biochem. Ztschr., 1910, XXV, 85; Jour. 
Path, and Bacteriol., 1910, XTV, 484; Browning and McKenzie, Diagnosis and Treatment 
of Syphilis, Philadelphia, 1912; Desmouliere, Presse Med., 1913, XXI, 898; Kolmer, Lau- 
baugh, Casselman and Williams, Arch. Int. Med., 1913, XII, 660; Thomas and Ivy, Jour. 
Am. Med. Assn., 1914, LXII, 363; Orkin, Berl. klin. Wchnschr., 1914, LI, 690; Thompson, 
Jour. Am. Med. Assn., 1914, LXII, 1458; Field, Ibid., 1620; Arch. Int. Med., 1914, XIII, 790; 
Judd, Jour. Am. Med. Assn., 1914, LXIII, 313; Owen and Snure, Jour. Mich. Med. Soc, 

1 914, XIII, 422; Interstate Med. Jour., 19 14, XXI, 12 81; Weston, Jour. Med. Research, 19 14, 
XXX, 377; McClure, Ibid., 455; Walker, Arch. Int. Med., 1914, XTV, 563; Hopkins and 
Zimmermann, Am. Jour. Med. Sc, 1914, CXLVHI, 390; Kolmer and Schamberg, Ibid., 1915, 
CXLIX, 365; Cornwall, New York Med. Jour., 1915, CI, 844; Henes, Jour. Am. Med. Assn., 

1915, LXIV, 1969. 

4 Ztschr. f. Chemotherap., Orig., 1912, I, 79. 

5 Jour. Exper. Med., 1913, XVIII, 75. 



62 2 DIAGNOSTIC METHODS. 

be used, although it is possible to use one-third of this dose providing very 
slight degrees of inhibition are disregarded and such tests called negative. 

This antigen has the great advantage that its antigenic and anti-comple- 
mentary properties are practically the same, although prepared from different 
hearts. The tests run through uniformly and always show the same degree of 
activity, making the interpretation of the results a matter of greater accuracy. 
This antigen may be recommended as a standard one and should be used in 
every test made, even though any number of antigens be used with it. 

c. Acetone-insoluble Extracts. 

As it has been shown that the alcoholic extracts of various organs contain 
variable amounts of "syphilitic antigen," Xoguchi 1 advocates the use of the 
alcohol and ether soluble but acetone insoluble extract of organs. This frac- 
tion contains lecithin and other phosphatids. 

Method of Noguchi. 

Extract a mashed paste of liver, heart, or kidney of man, ox, guinea-pig, 
rabbit, or dog with 10 parts of absolute alcohol at 37 C. for several days. 
Filter through filter-paper and collect the filtrate. The latter is then brought 
to dryness by evaporation with the aid of an electric fan or in a vacuum at 40 C. 
Take up the dried residue with ether and allow the turbid solution to stand in a 
covered dish over night in a cool place. Decant the clear ethereal solution into 
a clean beaker and concentrate by evaporating the most of the ether. Mix 
this concentrated ethereal solution with 10 volumes of pure acetone. Allow 
the precipitate to settle and decant off the supernatant fluid. This antigen is a 
light brownish precipitate which gradually becomes sticky on .exposure to the 
air. This product usually has no hemolytic action upon human red cells, but 
may occasionally show this property. Further, it may, also, show an anti- 
complementary action. Three-tenths gram of this acetone-insoluble fraction 
is dissolved in 1 c.c. of ether and is mixed with 9 c.c. of methyl alcohol. If any 
precipitate forms or is left undissolved, remove it by centrifugation. This stock 
solution remains unaltered for a long period. For the actual test an aqueous 
emulsion is prepared by mixing 1 c.c. of the stock solution with 9 c.c. of physio- 
logic salt solution. This produces a clear opalescent solution containing 0.3 
per cent, of the original lipoids. One-tenth c.c. of this emulsion is used in the 
test later described. This emulsion is kept on ice as it is unstable in this form. 2 

Antigen Paper. — Instead of keeping a stock solution of antigen as above 
described, Noguchi has introduced the use of paper impregnated with antigen 
for the test. These papers are especially serviceable to the general worker who 
may wish to employ this test but they do not hold their properties longer than 
three months. These are far inferior to the liquid antigen but may be used in 

1 Jour. Exper. Med., 1909, XI, 84; Serum Diagnosis of Syphilis, Philadelphia, 1912. 

2 Noguchi has recently (Jour. Am. Med. Assn., 1912, LVIII, 1163) prepared a specific 
antigen from cultures of the treponema pallidum. The most interesting finding, based upon 
comparative studies with this antigen and with the lipoid antigens, is that there is no indication, 
in cases of active syphilitic orchitis, of a sufficient amount of specific antibodies to bind comple- 
ment with the pallida antigen, although a strong positive Wassermann reaction obtains. See 
Craig and Nichols, Jour. Exper. Med., 1912, XVI, 336; Kolmer, Williams and Laubaugh, Jour. 
Med. Research, 1913, XXVIII, 345; Varney and Baeslack, Jour. Am. Med. Assn., 1913, LXI, 
754. 



THE BLOOD. 623 

an emergency. Weigh out about 1.2 grams of the sticky extract and dissolve in 
about 20 c.c. of ether. Have ten sheets of filter-paper, 10 by 10 cm. in dimen- 
sions, laid one upon the other in a clean glass dish. Pour over these the lipoid 
solution and saturate the paper evenly. Separate each sheet as quickly as 
possible and lay flat on a clean sheet of unbleached muslin. Within 10 min- 
utes the paper is ready for use. Before assigning the dimensions for each tube 
in the fixation test, the antigen paper should be titrated. This is done as 
follows: Cut the paper into equal width, say 5 mm., and use increasing lengths 
of this strip for standardization, starting with 1 mm., 2 mm., etc. The principle 
of this standardization will be discussed later. The strips may be marked in 
sections, each representing the required dimensions, and put into sealed tubes 
for preservation. 

The Syphilitic Antibody. 

The immune serum may be withdrawn from the patient by the method 
given under blood cultures. The blood, thus obtained, is allowed to coagulate 
in a large test-tube. The serum is then drawn off with a pipet into small test- 
tubes and inactivated by heating to 56 C. for one-half hour. 

In the writer's laboratory, the custom is to withdraw the blood from the 
median basilic vein by means of a pipet of about 20 c.c. capacity. One end of 
this pipet is attached to a rubber tube with glass mouthpiece, while the other 
end bears a steel or platinum-iridium needle attached with rubber tubing. Five 
to ten c.c. of blood are withdrawn and placed immediately in a small test-tube 
where it is allowed to clot. After about 20 minutes, the clot is separated from 
the walls of the tube and the tube is then centrifuged to obtain the clear serum. 
If the specimen is drawn by the physician and is to be forwarded to the labora- 
tory, it is essential that the cells be separated from the serum before the speci- 
men can be sent. Such specimens give reliable results, but if the cells are allowed 
to remain in contact with the serum for any length of time, so much hemolysis 
occurs that the test can not be accurately made by a distant laboratory. The 
serum is inactivated by heating to 5 6° C. for one-half hour either immediately 
after centrifugation or on receipt of the specimen. 

This inactivation is absolutely essential provided aqueous or alcoholic 
extracts are used as antigens, because these antigens contain various proteins, 
which are liable to give non-specific proteotropic fixation with active sera. 
Noguchi has shown that this property is lost if the serum be inactivated. More- 
over, with his antigen (acetone-insoluble lipoids) no such proteotropic fixation 
occurs, so that active sera may be employed in his test, although the inactivated 
serum works quite as well. 

Suspension of Red Cells. 
In the Wassermann test, the corpuscles to be hemolyzed are those of the 
sheep. The blood may be obtained from an abbatoir or sheep may be kept in 
the animal rooms of the laboratory and bled from the ear as desired. The blood 
is received directly in a more than equal amount of 1 per cent, sodium citrate 
solution in physiologic salt solution or it may be defibrinated by shaking with 
glass beads or by whipping with wires or bristles. It is then centrifuged and 



624 DIAGNOSTIC METHODS. 

the supernatant fluid drawn off. The cells are thoroughly mixed with physio- 
logic salt solution, again centrifuged and the clear fluid withdrawn. This 
process is repeated two or three times until the corpuscles are free from serum. 
The sedimented corpuscles are now drawn up with a graduated pipet and added 
to 19 volumes of salt solution. A 5 per cent, suspension is thus obtained, of 
which 1 c.c. is used in the test. If the centrifuge tube be calibrated, one may 
use this directly in making the suspension. Such suspensions may be used for 
two or three days, especially if kept in the ice box, but they should be discarded 
if any trace of hemolysis appears in the tube. 1 

Hemolytic Amboceptor. 

The hemolytic serum is obtained from rabbits which have been injected 
with washed sheep corpuscles. If the blood is received from the abbatoir, it is 
usually contaminated to such an extent that it must be sterilized by heating to 
6o° C. for one-half hour. The cells are obtained as above described and washed 
very thoroughly with salt solution. When free from serum, their volume is 
made up to that of the original defibrinated blood with physiologic salt solution. 
The immunization is best carried out by injecting a rabbit, intraperitoneally, 
with 2, 4, 8, and 12 c.c. of cells at intervals of 4 or 5 days. Ten days 
after the last injection a few c.c. of blood are obtained from an ear vein of the 
rabbit and the serum tested for its hemolytic effect upon sheep corpuscles. If 
it produces hemolysis in dilutions of 1 to 600 or more, the animal is bled from 
the carotid artery, the blood being collected in a series of sterile test-tubes. 
These are placed in the ice box, the blood allowed to coagulate and the serum is 
drawn off. This serum is then inactivated and put into small sterile tubes, 
which should be sealed and kept on ice. This material keeps well, but its 
strength must be determined at frequent intervals. It is wise to inject several 
animals at the same time, so that one may surely yield a satisfactory serum of 
high hemolytic power. 2 

Complement. 

Normal guinea-pig serum is used as complement in all modifications of this 
test. The animal is anesthetized and the blood drawn from the carotid, as 
above described, into sterile test-tubes in which it is allowed to coagulate. The 
serum is then drawn off with a sterile pipet, placed in small sterile test-tubes and 
kept on ice. 

As complement deteriorates rapidly (being of little use after 48 hours) the 
blood is, preferably, withdrawn by aspiration directly from the heart. 3 The 
animal will usually survive the loss of 5 to 10 c.c. of blood, providing the heart 
has not been badly lacerated by repeated punctures. After two weeks the 
animal may be used for a fresh supply of complement. It is wise to have quite 
a number of these animals on hand so that fresh material may constantly be 
available. It is absolutely unnecessary to sacrifice an animal each rime a few 

1 See Trossarello (Rif. Med., 1914, XXX, 95) also, Rous and Turner (Proc. Soc. Exper. 
Biol, and Med., 1915, XII, 122) for methods of preserving these blood cells. 

2 See Coca, Jour. Infect. Dis., 1915, XVII, 361; also, Morgenroth and Bieling, Biochem. 
Ztschr., 1915, LXVIII, 85. 

3 See Austin (Jour. Am. Med. Assn., 1914, LXII, 868) and Weston (Jour. Med. Research, 
1915, XXXII, 391) for methods of preserving complement. 



THE BLOOD. 625 

tests are to be made. When used complement is diluted 1 to 10 with physio- 
logic salt solution. 1 

Standardization of Reagents. 

This standardization must always be preliminary to the actual performance 
of the test. Everything depends upon the proper adjustment of the various 
factors entering into the test, so that especial care must be taken to find out just 
what quantities of each reagent to use. Without such precautions, little, if any, 
value can be attached to any report. 

Titration of Hemolytic Amboceptor. 

Prepare a series of dilutions of the inactivated serum beginning with 1 to 
300 and extending to 1 to 3,000. This may be readily accomplished by diluting 
0.1 c.c. of serum to 30 c.c. with physiologic salt solution and using this 1 to 300 
dilution as the basis of further dilutions. Arrange a series of small test-tubes 
and place in each 0.25 c.c. of these diluted sera, beginning with the highest con- 
centration and extending to the lowest. Now add 0.25 c.c. of a 1 to 10 dilution 
of complement and 0.25 c.c. of a 5 per cent, suspension of sheep corpuscles to 
each tube. Bring the total volume up to 1.25 c.c. with physiologic salt solution. 
All tubes are placed in the incubator at 37 C. for two hours and are then allowed 
to stand over night in the ice box. The highest dilution (smallest amount of 
amboceptor) in which complete solution of the cells occurs is taken as the 
strength of the serum. This is called one amboceptor unit. In performing the 
Wassermann test, two units of amboceptor are used. The titer of the ambocep- 
tor remains fairly constant, but it is wise invariably to determine its value when 
fresh complement is to be used, owing to the marked influence of these factors 
on one another. 

Instead of Using the above method of standardization, one may resort to 
the drop method of Landsteiner, Muller and Poetzl, which simplifies the technic 
but, in so doing, impairs the accuracy of the result, in the writer's opinion, as 
the drops from different capillary or other pipets are seldom of the same size. 
By this method one determines what dilution of amboceptor in 1 drop dose will 
hemolyze in one-half hour, in an incubator, 1 drop of 50 per cent, suspension of 
sheep's corpuscles with 1 drop of complement, 10 drops of salt solution being 
added as diluent. Twice the strength of the titer is used in the test. 

Titration of Complement. 

Having obtained the value of the amboceptor unit, one may use decreasing 
amounts of complement with one unit of amboceptor in the same way as in the 
above titration, the dilution of the complement running from 1 to 2 to 1 to 20. 
Complement is usually used in a dilution of 1 to 10, 1 c.c. being employed in the 
test. This is, however, open to objections as it is not absolutely quantitative. 
Noguchi has pointed out the fact that variation in the amount of complement 
used influences the amount of amboceptor. "If less than one unit of ambo- 

1 See Tissot (Compt. rend. Acad, de Sc, 1914, CLVIII, i525);Thorsch (Biochem. Ztschr., 
1915, LXVTII, 67); Ottenberg, Reuben andFrazier (Jour. Infect. Dis., 1915, XVI, 119) for stud- 
ies of the nature of complement. Cole (Cleveland Med. Jour., 1915, XIV, 273) shows the 
presence of anti-sheep amboceptor in guinea-pig's blood. 
40 



626 DIAGNOSTIC. METHODS. 

ceptor is used hemolysis will always be incomplete, even with more than one unit 
of complement. Likewise, if with one amboceptor unit there is combined less 
than one unit of complement, hemolysis can not be complete. If with more than 
one unit of amboceptor there be used less than one unit of complement, hemoly- 
sis may be complete or incomplete according to the relative amounts of each 
factor used. 1 In the presence of many units of amboceptor hemolysis may be 
complete when but a small fraction of the complement unit is present." 

Titration of Antigen. 

The degree of dilution in which the antigen is to be used must be deter- 
mined for each extract by testing it both against normal and syphilitic sera. 
Its concentration must be such that it does not prevent hemolysis with normal 
sera, while it will promptly inhibit hemolysis in presence of complement with 
syphilitic sera. 

Set up two parallel series of tubes. Into each tube of one series place 0.25 
c.c. of a 1 to 5 dilution of known syphilitic serum and into each' tube of the 
second series place 0.25 c.c. of a 1 to 5 dilution of normal serum. Dilutions of 
the antigen are now made, with physiologic salt solution as the diluent, begin- 
ning with 1 to 1 and running up to 1 to 20 or higher if necessary. Into the first 
tubes of the above series place 0.25 c.c. of the first antigen dilution, into the 
second tubes of each series place the second dilutions and so on until all are 
accounted for. To all of the above tubes add 0.25 c.c. of complement (1 to 10) 
and incubate at 37 C. for one hour. Now add 0.25 c.c. of hemolytic ambocep- 
tor in twice the maximum dilution found to cause complete hemolysis (two 
units) and 0.25 c.c. of a 5 per cent, suspension of sheep corpuscles. Place in an 
incubator for two hours and then in ice box over night. No hemolysis should 
be observed in the tubes containing syphilitic serum except in those containing 
the higher dilutions, while in the tubes with normal serum hemolysis will occur 
in all tubes except those with the lower dilutions of antigen. Hemolysis in the 
tubes with syphilitic sera is due to the normal hemolytic power of the antigen, 
while the lack of hemolysis in the tubes with normal sera is due to the anti- 
complementary action of the antigen. In carrying out the Wassermann test, 
antigen is used in one-half the lowest dilution (largest amount of antigen) which 
gives complete hemolysis with normal sera, this being taken as one antigen unit. 
Thus if a dilution of 1 to 5 gave complete hemolysis, a dilution of 1 to 10 would 
be used in the actual test. 

If one uses the drop method, as mentioned above, 1 drop of antigen (undi- 
luted) and 1 drop of complement (undiluted) are mixed with 10 drops of salt 
solution. The mixture is incubated one-half hour and 1 drop of amboceptor 
and 1 drop of a 50 per cent, suspension of corpuscles are added. Hemolysis 
should occur within one-half hour. If it does not, anticomplementary action 
is marked and less antigen must be employed in the test. 

Having determined the strength of the antigen, a portion of the stock solu- 
tion is diluted with salt solution so that 1 c.c. equals one unit. In testing the 

^ * See Kromayer and Trinchese, Med. Klin., 1912, VIII, 404 and 1670, who use an absolute 
minimum of complement in their "refined" Wassermann test; also. Thompson, Arch. Int. 
Med., 1914, XIII, 904. 



THE BLOOD. 627 

antigenic properties of this antigen, one may use it with a large number of posi- 
tive and normal controls, or, preferably, run it in parallel series with an antigen 
of known value. If the results agree, it may then be substituted for the old 
antigen. The titer being known, the antigen may be used for subsequent 
tests without redetermination of this value. 

Technic of the Test. 
Original Wassermann Method. 

All of the glassware used in the test must be kept thoroughly clean and dry, 
but need not be sterile. The pipets, which should be both graduated to 0.01 
and 0.1 c.c., should, preferably, be used with only one reagent. If they are 
employed with others, they must be thoroughly rinsed in salt solution between 
such applications. After using, wash them well in salt solution (never with 
soap and water), allow them to stand in a tall cylinder of distilled water for a 
few hours and dry them in an oven. The test-tubes used in this test are about 
14 cm. in length by 1.5 cm. in diameter, while the somewhat smaller ones (1 by 
10 cm.) may be employed in the Noguchi modification. 

Before actually performing the test, the reagents are prepared and stand- 
ardized as previously described or the amboceptor may be titrated at the begin- 
ning of the test, as it is not added until the preliminary incubation of the serum 
with the antigen and complement. The sera of the patient and of the known 
positive and normal controls must all be inactivated by heating to 56 C. for 
one-half hour. 1 The complement is diluted 1 to 10 with physiologic salt solu- 
tion and the suspension of washed sheep corpuscles prepared. When these 
preliminaries are attended to one may proceed with the test itself. 

Arrange a double row of test-tubes, the front tube of each set to receive the 
antigen and the back row the control tube without antigen. One set of tubes 
must be placed for the unknown serum, one set for the positive and one set for 
the normal control; while other controls are arranged as spoken of later. 

1. Into both tubes of each set place 0.2 c.c. of the serum to be tested. 

2. To all tubes, add 1 c.c. of complement (diluted 1 to 10). 

3. To tubes in front row only, add 0.2 c.c. of antigen (aqueous extract of 
syphilitic liver). 

Make up volume of each tube to 3 c.c. with salt solution, mix contents by 
shaking and incubate at 37 C. for one hour. 

4. Add to each tube 1 c.c. of antisheep amboceptor (two units). 

5. Add to each tube 1 c.c. of 5 per cent, suspension of sheep corpuscles. 
Mix the contents of the tube thoroughly, incubate at 37 C. for two hours 

and place the tubes in the ice box over night. At the end of this period the 
results are read. 

Controls. 

1. Positive syphilitic serum. To be run as above. 

2. Known negative serum. To be run as above. 

1 Gramenitzki (Biochem. Ztschr., 1912, XXXVIII, 501) and Fenyvessy (Ibid., 1912, XL, 
353) have shown that if the serum be not heated to complete inactivation, the complement 
is restored on allowing to stand for some time. 



628 DIAGNOSTIC METHODS. 

3. A tube containing complement and two units of antigen, to prove that 
no anticomplementary action of the antigen obtains. 

4. A tube containing complement, to show that the hemolytic system is active. 

5. A tube containing antigen alone, to show absence of lytic power of antigen. 
All controls are brought up to 3 c.c, incubated and, then, amboceptor and 

sheep corpuscles are added as above. 

If the test is properly conducted, hemolysis will occur in every control tube 
excepting in the tube containing syphilitic serum and syphilitic antigen (the 
positive control) and in the tube of control 5. In the series of unknown sera, 
no hemolysis will occur in the tubes containing syphilitic serum. This lack of 
hemolysis, partial or complete, is, of course, a positive reaction. 

Citron 1 advises the use of two tubes for serum ancl antigen, one tube con- 
taining 0.2 c.c. of serum and 0.2 c.c. of antigen as given above, while the other 
tube contains 0.1 c.c. of serum and 0.1 c.c. of antigen. In this way the results 
may be represented somewhat more quantitatively, as the degree of inhibition 
of hemolysis varies according to the amount of syphilitic antibody present. If 
this amount be large, complete inhibition occurs in the tube containing 0.1 c.c. 
of serum and 0.1 c.c. of antigen as well as in tube No. 1, the result being ex- 
pressed as + + + + . If inhibition is incomplete in tube 2 but complete in tube 
1, the result is + + + . Both of these degrees are strongly positive reactions. 
If tube 2 is completely hemolyzed while tube 1 is completely inhibited, the result 
is + + . Incomplete inhibition in tube 1 and complete hemolysis in tube 2 is 
expressed as + . These two latter results are classed as weakly positive reac- 
tions. If tube 1 shows doubtful inhibition while tube 2 shows complete hemo- 
lysis, the result is +. Complete hemolysis in both tubes is, of course, a negative 
result. 2 

Noguchi's Modification. 

This is not to be confused with Noguchi's test in which a human hemolytic 
system is used. The modification here suggested uses a different antigen and 
different quantities of the same reagents as employed by Wassermann. This 
is the method, followed in the writer's laboratory, when the sheep hemolytic 
system is used, a control being run with Sachs' antigen or a specific watery 
extract as antigen in cases showing affections of the central nervous system. 

1. Set up the double row of tubes as in the preceding test using 0.2 and 0.1 
c.c. of serum to be tested. 

2. To all tubes add 0.1 c.c. of an undiluted complement. 

3. To front row only, add 0.1 c.c. of acetone-insoluble antigen. 

Make up volume of each tube to 1.5 c.c. with physiologic salt solution. 
Mix contents thoroughly and incubate at 37 C. for one hour. 

4. Add to each tube 1 c.c. of antisheep amboceptor (two units). 

1 Handbook of Kraus and Levaditi, 1908, II, 1105. 

2 Ivy (Jour. Am. Med. Assn., 19 12, LIX, 432) advocates the use of the Duboscq colorimeter 
as a means of estimating the exact degree of hemolysis, the control tube of each case being 
taken as the standard of 100 per cent. Ninety per cent, and over of hemolysis is regarded as 
negative. See Kaplan (New York Med. Jour., 1913, XCVII, 1172; Ibid., 1913, XCVIII, 157); 
and Kaplan and McClelland (Ibid., 1012) for the determination of amino nitrogen as a 
control. 



THE BLOOD. 629 

5. Add to each tube 0.5 c.c. of a 10 per cent, suspension of sheep corpuscles. 

The total volume is now 3 c.c. Mix contents thoroughly and incubate at 
37 C. for two hours. Allow to stand four to six hours at room temperature and 
read results. The interpretation is as given above. 

Drop Method. 

This is the method of Landsteiner, Miiller and Poetzl and is frequently 
employed. The writer can not convince himself that this method is an accurate 
one owing to the possible variations in size of the drops, which are delivered 
from a capillary pipet. 

1. Set up the double row of tubes as in the preceding tests and add to each 
10 drops of physiologic salt solution. 

2. Add 1 drop of serum to each of the two tubes used for the sera tested. 
'3. To front row only, add 2 drops of antigen. 

4. To all tubes add 1 drop of complement. 

Mix thoroughly and incubate for one hour at 37 C. 

5. Add 1 drop (two units) of standardized antisheep amboceptor to each tube. 

6. Add 1 drop of a 50 per cent, suspension of sheep corpuscles to each tube. 
Incubate for two hours. Allow to stand at room temperature and read 

results. The method of interpretation is as above. 1 

Method of Noguchi. 2 
It has been found that human serum contains a variable amount of natural 
antisheep amboceptor. 3 This, of course, increases the effect of the immune 
antisheep amboceptor used in the tests previously described. One may, there- 
fore, obtain a negative Wassermann test under these circumstances even though 
a large amount of complement is fixed. Noguchi has overcome this possible 
error by introduction of a human hemolytic system to replace the sheep hemo- 
lytic system of the original test. He employs, also, a smaller amount of reagents 
and uses either fresh or inactivated serum of the patient. If preferred, one may 
use larger quantities of patients , serum, than outlined by Noguchi; if so, the 
reagents are increased in the same proportion. This method of Noguchi is the 
one preferred by the writer and is the one followed as a routine in his laboratory, 
as it has been found somewhat more reliable than the original Wassermann test 
in obscure cases. 4 Sachs' antigen is used in every test. 

Collection of Serum. 

Noguchi obtains the patient's blood from a puncture on the ventral side of 
the last joint of the middle finger. The finger may be compressed by squeezing 
before the puncture and, afterward, massaged to press out the blood, this being 
collected in small tubes drawn out into capillary points. In the writer's labora- 
tory the blood is obtained as given on page 623. 

1 See Mefford, IUinois Med. Jour., 1914, XXVI, 428. 

2 Jour. Exper. Med., 1909, XI, 392; Serum Diagnosis of Syphilis, Phila., 1912. 

3 See Dexter and Cummer, Arch. Int. Med., 1912, IX, 605; also, Bailey, Ibid., 551. 

4 See Coca and L'Esperance (Arch. Int. Med., 1913, XI, 84) for a slight modification of 
the Noguchi technic. See, also, Miller, Interstate Med. Jour., 1913, XX, 145; Thompson, 
Arch. Int. Med., 1913, XI, 512. See Cecil and Lamb (Arch. Int. Med., 1913, XI, 249) for the 
value of this test in testing the serum of cadavers. 



630 



DIAGNOSTIC METHODS. 



Corpuscle Suspension. 

The suspension may be prepared with the blood of the patient or with that 
of the examiner. If with that of the former, it is especially important that every 
trace of serum be removed by careful washing. The standard amount of cor- 
puscle suspension is 1 c.c. of a 1 per cent, or 0.1 c.c. of a 10 per cent, suspension 
for each tube. If the patient's blood is to be used, enough may be obtained for 
many tests as follows: Fill a graduated centrifuge tube to 9 c.c. with 2 per cent, 
sodium citrate in physiological salt solution. Allow the blood to drop in until 
it fills the tube to 10 c.c. Mix and centrifuge. Pour off the supernatant fluid 
and fill up to 10 c.c. with fresh salt solution. Mix thoroughly and again centri- 
fuge. This may be repeated two or three times, in order to insure removal of 
every trace of serum. The deposited corpuscles may now be suspended either 
in 100 c.c. of salt solution (making a 1 per cent, suspension) or in 10 c.c. (giving 
a 10 per cent, suspension). This procedure, while thoroughly reliable, is not 
followed by the writer. It is our custom to obtain the corpuscles from a punc- 
ture of the finger of the worker, allowing 3 drops of blood to fall into 12 c.c. of 
salt solution in a centrifuge tube. This gives, approximately, a 1 per cent, 
suspension. The washing is done as above. This latter method enables us to 
obtain fresh suspensions whenever desired. 

Hemolytic Amboceptor. 

This is prepared, as previously outlined, by injecting a rabbit, intraperi- 
toneally, with increasing doses of washed human corpuscles, instead of with 
sheep corpuscles as in the Wassermann method. After the animal is immu- 
nized, the blood is collected and allowed to coagulate. The serum is then with- 
drawn and titrated as previously described. 

Amboceptor Paper. — This has been introduced by Noguchi to simplify the 
test and to enable those not in close touch with clinical laboratory facilities to 
perform the test. While this paper may be used, the writer advises those doing 
any number of these tests to place their reliance more in the liquid amboceptor. 
The serum, obtained as above, is poured over sheets of filter-paper (Schleicher 
and SchulPs No. 597), cut into squares of 10 x 10 cm. Allow all the sheets to 
become evenly wet and absorb the excess with another sheet of paper. These 
are then dried at room temperature by placing each square separately upon a 
clean sheet of unbleached muslin. When dry, the sheets are cut into strips 5 
mm. wide and are then standardized as follows : Take a series of tubes con- 
taining 1 c.c. of the 1 per cent, erythrocyte suspension and add to each tube 
0.02 c.c. (as one unit) of complement. Now add the strips in increasing 
lengths, as 1 mm., 2 mm., 3 mm., etc., and incubate for two hours. The 
shortest strip causing complete hemolysis in this time represents one unit of 
amboceptor. These strips are then marked into sections of twice this length 
(two units) and cut off when the actual test is to be made. The papers should 
be kept dry and sealed. The papers on the market are quite variable and are 
not always to be relied upon. As previously stated, these papers should be 
used only when the liquid amboceptor is not at hand. 



THE BLOOD. 



63I 



ORIGINAL WASSERMANN TEST. 





S 


ti -o* 




A d 




CO 

CU 








2 
£ 

d 

a 


omplemer 
uinea-pig 
um 1 to 1 


d 

cu 

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Antisheep 
mbocepto 
two units 


ve per cen 
spension < 
ep corpus 


CO 




Hemolysis 




tn 


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Unknown serum 


c.c. 
0.2 


c.c. 

I 


c.c. 

0.2 


-d jg-a 

Cj d dj 


C.C. 

I 


C.C. 

I 




Depends on sera 


Unknown serum 


0.2 


I 




£ S § 


1 


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*£•§> 


Complete. 


Positive control 


0.2 


I 


0.2 


0.«o 


I 


I 


ts c 


None. 


Positive control 


0.2 


I 




a^ ^ 


I 


I 


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Complete 


Negative control 


0.2 


I 


0.2 


3 


I 


I 


His 


Complete. 


Negative control 


0.2 


I 




es 


I 


I 


^< 


Complete. 


Anticomplementary 




I 


0.4 


Dh'-S 


I 


I 




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control. 


















Hemolytic control 




I 




44 M 


I 


I 


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None. 


Antigenic control 






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NOGUCHI'S MODIFICATION (SHEEP HEMOLYTIC SYSTEM). 




s 


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p 








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serum undil 


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ne hour 


Ten per cen 

suspension c 

sheep corpusc 


Antisheep 
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n 


Hemolysis 




c.c. 


c.c. 


c.c. 


c.c. 


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Unknown serum 


0.2 


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Unknown serum 


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Complete. 


Unknown serum 


O.I 


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O.I 


psrf 


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Unknown serum 


O.I 


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Complete. 


Positive control 


O.I 


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O.I 


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None. 


Positive control 


O.I 


0.1 




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Negative control 


0.2 


0.1 


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0.5 


1 


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Complete. 


Negative control 


0.2 


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Anticomplementary 




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0.2 


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control. 








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NOGUCHI'S M( 


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a 

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Complement 

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serum 1 to 40) 


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suspension of 

human corpuscles 


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Unknown serum 


0.02 


O.I 


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1 


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2 units 


2-S 


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Unknown serum ...... 


0.02 


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1 


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£^3 


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Positive control 


0.02 


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Positive control .... 


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Negative control 


0.02 


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Negative control 


0.02 


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1 


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Anticomplem e n t a r y 




O.I 


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3 


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control. 
















Hemolytic control 




O.I 




1 




2 units 


Complete. 







632 DIAGNOSTIC METHODS. 

Complement. 

Guinea-pig serum is used as complement in this system as in the others. 
The dilution is 40 per cent, made by mixing 1 c.c. of complement with 1 1/2 c.c. 
of physiologic salt solution. The method of obtaining it has been given above. 
Complement paper may be prepared but it is very unsatisfactory and should not 
be used if the liquid form can possibly be obtained. 

Antigen. 

The antigen, used in this system, is prepared as previously described (p. 
622). The stock solution is a 3 per cent, methyl alcohol solution of the acetone- 
insoluble lipoids. From this a 0.3 per cent, emulsion is prepared for the test by 
mixing 1 c.c. of this stock solution with 9 c.c. of physiologic salt solution. It 
seems advisable always to control this antigen by running a control test with a 
watery extract of specific organs, and of Sachs' antigen, especially if an affec- 
tion of the central nervous system obtains. 

Technic. 

1. Arrange the double row of tubes with same controls as in the other tests. 
Place in all tubes 1 capillary drop (0.02 c.c.) of the fresh serum to be tested. 
If the serum has been inactivated use 4 drops (0.08 c.c). One may use 0.2 c.c. 
of fresh (not inactivated) cerebrospinal fluid. 

2. Add to each tube 0.1 c.c. of 40 per cent, complement, or in a great 
emergency two units of complement paper. 

3. To the tubes in front row of each set, add 0.1 c.c. of antigen emulsion. 

4. To both tubes of each set, add 1 c.c. of 1 per cent, washed human corpuscles. 
Mix thoroughly and incubate for one hour at 37 G. 

5. Add to each tube 2 units of antihuman amboceptor in liquid or paper form. 
Mix and incubate for two hours at 37 C. Allow the tubes to stand at 

room temperature for a few hours and read results. 

The results of this test are interpreted as in the preceding ones. This 
human system is more adjustable and reliable than is the sheep system. Nogu- 
chi has pointed out certain variable factors which may obtain and which may 
require a proper adjustment to insure proper results. These variables are, it 
is to be understood, greater in the sheep system, and are far less easily controlled. 
I quote directly from Noguchi. (1) One sometimes meets with instances in 
which hemolysis is complete within 10 to 20 minutes, and in which the positive 
control tubes with antigen undergo, sooner or later, gradual hemolysis. Such 
rapid progress of hemolysis at first mentioned is a sign of imperfect reaction. 
If the test is properly made, hemolysis proceeds gradually, and is complete in 
the water-bath within half an hour or thereabout. The causes of this acceler- 
ated hemolytic process are either an abnormally weak resistance of the blood 
corpuscles, or an exceptionally high activity or insensitiveness to fixation of the 
complement employed; or it may be the result of all these acting together. 1 It 
happens occasionally that the serum of certain guinea pigs contains an abnor- 
mally active complement. In order to establish this point, and thus to remove 
this source of error, one has only to make the test with a quantity of comple- 
1 See Bailey, Jour. Exper. Med., 19 12, XV, 470. 



THE BLOOD. 633 

merit which corresponds exactly to two complement units. (2) There are 
sometimes encountered instances in which hemolysis remains incomplete even 
in the control tubes in which there is no antigen. Here the causes of the imper- 
fect reaction are found either in the weakness of the complement, or the ambo- 
ceptor used, or both. 1 Usually the cause is the weakness of the complement, 
which, owing to its great lability, is likely to deteriorate. The activity of the 
amboceptor is far less subject to external influences which bring about its 
deterioration, and it is therefore extremely rare to find that the imperfection in 
the reaction arises from this source. In testing several specimens of serum at 
one time it happens occasionally that some specimens are slower in completing 
the hemolytic reaction than others. The cause of this slowness is not present 
in the complement or amboceptor, but in the specimens themselves. 2 In such 
cases the specimens are found to contain anticomplementary substances which 
react with and reduce the activity of the complement. To remove this source 
of error, it is necessary to heat the serum to 55 C. for 20 minutes and use four 
drops for the test. The difficulty may be obviated in some cases by collecting 
specimens of serum to be tested just before meal-time because the anticomple- 
mentary substance is closely associated with the absorption of the chyle into the 
circulation soon after the meal. (3) The quality and quantity of the antigen 
can also be sources of error. If one uses poor antigen, either there will be no 
positive reaction at all, or weak positive reactions will be entirely overlooked. 
If, on the other hand, an excessive amount of unfractionated crude antigen is 
employed, certain nonspecific weak reactions may become manifest, or a false 
positive reaction even may be obtained, as the result of the action of anticom- 
plementary substances sometimes contained in preparations of the antigen. 
These sources of error can be entirely excluded by choosing an antigen that has 
been carefully prepared and standardized. 3 

Diagnostic Value of the Wassermann Test. 

As stated above, the Wassermann reaction, or any of its modifications, is 
not a specific immunity reaction. While the antibody is, presumably, syphilitic 
in origin, the antigen is a physiologic lipoid and not a product of the treponema 

1 See Barratt, Jour. Path, and Bacterid., 1912, XVI, 363. 

2 See Bronfenbrenner and Noguchi, Jour. Exper. Med., 1912, XV, 598 and 625. 

3 For discussions of many obscure points regarding the nature of the reagents and the 
technic of the test see Addis, Jour. Infect. Dis., 1912, X, 200; Dick, Ibid., 1913, XII, in; 
Lippmann and Plesch, Ztschr. f. Immunitatsforsch., 1913, XVII, 548; Baumann, Illinois Med. 
Jour., 1913, XXrV, 117; Burdick, Colorado Med., 1913, X, 135; Kolmer and Casselman, Jour. 
Med. Research, 1913, XXVIII, 369; Surface and Routt, Ibid., 441; Cummins, Ibid., 1913, 
XXTX, 23; Weil, Biochem. Ztschr., 1913, XLVIII, 347; Stillians, Jour. Cutan. Dis., 1913, 
XXXI, 316; von Gierke, Deutsch. med Wchnschr., 1913, XXXIX, 692; Rabinowits'ch, Ibid., 
1210; Kolmer and Williams, Jour. Infect. Dis., 1913, XIII, 96; Spat, Biochem. Ztschr., 1913, 
LVI, 21. Kapsenberg, Nederl. Tijdschr. v. Geneesk., 19 13, II, 1007; Wassermann and 
Lange, Kolle and Wassermann's Handb., 1913, VII, 951; Lesser, Munch. Med. Wchnschr., 
1914, LXI, 70; Langer, Deutsch. med. Wchnschr., 1914, XL, 274; Olmstead, Med. Record, 

1914, LXXXV, 341; Thiele and Embleton, Lancet, 1914, I, 526; Mcintosh and Fildes, Ibid., 
860; Matzenauer and Hesse, Wien, klin. Wchnschr., 1914, XXVII, 319; Sternberg, Ibid., 545; 
Klein and Frankel, Munch, med. Wchnschr., 1914, LXI, 651; Wechselmann, Berl. klin. 
Wchnschr., 1914, LI, 304; Blumenthal, Ibid., 1316; Nathan, Ibid., 1934; Stone, New York 
Med. Jour., 1914, XCLX, 1242; Grulee, Am. Jour. Med. Sc, 1914, CXLVIII, 688; Lyon and 
Eiman, Ibid., 885; Stephens, Arch. Diag., 1915, VIII, 126; Schlesinger, Med. Record, 1915, 
LXXXVII, 61; Bana, Brit. Med. Jour., 1915, I, 587; Stitt and Clark, U. S. Naval Med. Bull., 

1915, VIII, 410; Clark, Ibid., 411. 



634 DIAGNOSTIC METHODS. 

pallidum. In other words, the complement fixation test, as applied to the 
diagnosis of syphilis, is not the result of an interaction between homologous 
(specific) antigen and antibody. Nevertheless, we find that the reaction does 
have a certain specificity from the diagnostic, if not from the biologic, standpoint. 

As a general rule it may be stated, at the outset, that a positive Wassermann 
test, all proper precautions and controls being made, indicates syphilis in 
practically all cases. It is true that certain nonspecific conditions, especially 
leprosy and, occasionally, scarlet fever, give positive reactions, but the large 
majority of cases of other diseases, reported as giving positive results, are those 
in which syphilis has not been absolutely excluded. In such cases the syphil- 
itic infection may be latent instead of the active cause of the trouble at the time 
of making the test, yet it must be reckoned with in deciding as to the value of 
this test from the diagnostic point of view. x 

On the other hand, it is to be distinctly understood that a negative Wasser- 
mann test does not exclude syphilis, a fact that must be taken into consideration 
when the question of marriage is involved. Such cases must be judged largely 
by the clinical manifestations and history rather than upon the negative char- 
acter of this test. Varying percentages of positive results in known syphilitic and 
parasyphilitic conditions have been reported by various workers. Some of the 
more enthusiastic ones claim that a negative Wassermann reaction means that 
the case is not syphilitic. This view, however, must be regarded as untenable 
in the face of the large numbers of negative results in the literature and in the 
daily experience of those working in this field. The following table, compiled 
from Boas 2 and Noguchi 3 shows the reported cases of syphilis with the percent- 
ages of positive results. 4 

1 See Kolmer, Jour. Exper. Med., 1911, XIV, 235; Field (Jour. Am. Med. Assn., 1912, 
LVIII, 1681) has called attention to the occurrence of positive Wassermann tests in cases of 
lead poisoning. Hilgermann, Deutsch. med. Wchnschr., 1912, XXXVIII, 118; Gibson, Aus- 
tralasian Med. Gaz., 1913, XXXIII, 300. For other diseases showing a Wassermann see Jako- 
bovics, Jahrb. f. Kinderhkde., 1914, XXIX, 215; Fletcher, Lancet, 1914, I, 1677; Butier, U. S. 
Naval Med. Bull., 1915, IX, 51; Hesse, Wien. klin. Wchnschr., 1915, XXVIII, 62; Sutherland 
and Mitra, Indian Jour. Med. Research, 1915, II, 984. 

2 Die Wassermannsche Reaktion., Berlin, 1913. 

3 Serum Diagnosis of Syphilis, Philadelphia, 19 12. 

4 See Lucas, Am. Jour. Dis. Child., 1912, III, 259; Churchill, Ibid., 363; Kaplan, Med. 
Record, 1912, LXXXI, 1132; Bates, Arch. Int. Med., 1912, X, 470; De Buys, Am. Jour. Dis. 
Child., 1913, V, 65; Milne, Am. Jour. Med. Sc, 1913, CXLV, 197; Wolbarst, New York Med. 
Jour., 1913, XCVII, 378; Grindon, Davis, Greiner and Weiss, Interstate Med. Jour., 1913, 
XX, 221; Sarateanu and Velican, Monatsschr. f. Geburtsh. u. Gynak., 1913, XXXVII, 89; 
Balcarek, Med. Klin., 1913, IX, 1552; Eliasberg, Deutsch. Ztschr. f. Chir., 1913, CXXTV, 113; 
Blackfan, Nicholson and White, Am. Jour. Dis. Child., 1913, VI, 162; Holt, Ibid., 166; Rich- 
ards, Jour. Am. Med. Assn., 1913, LX, 1139; Kaplan, New York Med. Jour., 1913, XCVIII, 
308; Mcllroy, Watson and Mcllroy, Brit. Med. Jour., 1913, II, 1002; Heidingsfeld, Jour. Am. 
Med. Jour., 1913, LXI, 1598; Mensi, Gazz. d. osp., 1913, XXXIV, 1032; Post, Boston, Med. 
and Surg. Jour., 1913, CLXIX, 777; Kaplan, Jour. Am. Med. Assn., 1913, LXI, 2214; 
Emery, Lancet, 1914, I, 223; Philippson, PoHclinico, 1914, XXI, 20; Russo, Riv. osped., 
1914, IV, 173; Descomps, Grece Med., 1914, XV, 37; Lesser and Klages, Deutsch. med. 
Wchnschr., 1914, XL, 1309; White, Therap. Gaz., 1914, XXXVIII, 307; MacKinney, Ann. of 
Surg., 1914, LX, 309; Bahr, Illinois Med. Jour., 1914, XXV, 245; Corbus, Ibid., 1914, XXVI, 
112; Craig, Jour. Am. Med. Assn., 1914, LXII, 1232; Thomas and Ivy, Am. Jour. Med. Sc, 

1914, CXLVIII, 55; Southard, Boston Med. and Surg. Jour., 1914, CLXX, 947; Stone, Med. 
Record, 1914, LXXXVI, 545 ; Fordyce, New York Med. Jour., 1914, C, 597; Craig, Am. Jour. 
Med. Sc, 1915, CXLIX, 41; Krauss, Biochem. Ztschr., 1915, LXVUI, 48; Pontano, Policlinico, 

1915, XXII, 41, 77 and 113; von Gonzenbach, Cor.-Bl. f. Schweiz. Aerzte, 1915, XLV, 161 and 
225; Wolbarst, Interstate Med. Jour., 1915, XXII, 109; Gradwohl, Southern Med. Jour., 1915, 



THE BLOOD. 



635 



Condition 


Number of cases 


Positive results 


Primary syphilis 


974 

2,762 

830 

1,233 

861 

125 

64 

498 

360 


56.5 per cent. 


Secondary syphilis 


88 . per cent. 
80.0 per cent. 


Tertiary syphilis 


Early latent syphilis 


51 .0 per cent. 


Late latent syphilis 


47 .0 per cent. 


Congenital syphilis 


94 . 5 per cent. 
47 . 6 per cent. 
88 . 1 per cent. 


Cerebrospinal syphilis 


General paralysis 


Tabes dorsalis 


70.0 per cent. 





Considerable discussion has arisen in the literature regarding the relative 
merits of the Wassermann and Noguchi systems. As a general rule it is found, 
by one running these two systems in parallel, that the positive results are some- 
what higher with the Noguchi system. The following table, taken from 
Noguchi, gives the figures for the cases reported, in which the two tests were 
made upon the same serum. 



Condition 



Number of 


Wassermann 


Noguchi 


cases 


positive 


positive 


208 


88 per cent. 


94 per cent. 


669 


92 per cent. 


98 per cent. 


455 


74 per cent. 


83 per cent. 


305 


54 per cent. 


68 per cent. 


79 


98 per cent. 


98 per cent. 


55 


73 per cent. 


80 per cent. 



Primary syphilis 

Secondary syphilis . . . 

Tertiary syphilis 

Latent syphilis 

Congenital syphilis . . . 
Cerebrospinal syphilis 



Effect of Treatment on the Reaction. 

Under the influence of a vigorous course of mercurials, a positive Wasser- 
mann test may be made to disappear. This must, however, not be interpreted 
as meaning that the syphilitic infection has been cured. It is almost daily 
observed by those doing large numbers of these tests that a patient, having the 
test made while under active treatment with mercury, will frequently give a 
negative test; while the same patient may show a positive reaction if the mercury 
be withdrawn for two or three weeks prior to the test. On the other hand, we 
occasionally find cases in which the treatment has resulted in a positive Wasser- 
mann, prior tests having been negative. These facts must be borne in mind by 
those who may attempt to establish a diagnosis without full knowledge of the 
conditions under which the test is being made. Mercurial treatment may render 
the infection latent, so that a negative reaction may obtain until later active 
manifestations appear and become associated with a positive test. This 
variability has been shown by Craig, 1 who reports 52 cases treated, with 32 

VIII, 477; Robertson and Klauder, Jour. Am. Med. Assn., 1915, LXTV, 199; Keyes, Ibid., 
804; Vedder and Hough, Ibid., 972; Weisenburg, Ibid., 975; Haberman, Ibid., 1141; Hei- 
mann, Ibib., 1463; Wile and Stokes, Ibid., 1465; Glomset, Ibid., 1915, LXV, 682; Uhle and 
MacKinney, Ibid., 863. 

1 Jour. Exper. Med., 1910, XII, 726; Arch. Int. Med., 1911, VIII, 395. Craig and Nichols 
(Jour. Am. Med. Assn., 1911, LVII, 474) and Craig (Ibid., 1913, LX, 565) call attention to 
the influence of alcohol in converting a positive test into a negative one within 24 hours. 



636 DIAGNOSTIC METHODS. 

positives and 20 negatives after treatment ranging from two weeks to twelve 
years. Patients should not have had any mercurial treatment for at least two 
to three weeks prior to the test if any reliance at all is to be placed upon it. 
Patients well treated give negative reactions, while those receiving inadequate 
treatment, no matter for how long a period, show positive reults. The 
reaction is, unquestionably, affected by mercurial treatment, but some cases 
persist in giving a positive reaction in spite of treatment. The reaction may 
return shortly after treatment, while it is not, by any means, settled that the 
disappearance of a reaction justifies one in stopping treatment. 

After treatment with "606" (salvarsan, dioxy-dimino-arseno benzol), 
the results are variable. In promptly cured cases, a positive reaction may dis- 
appear within two weeks or it may requie four to five weeks to obtain a negative 
result. A positive reaction may persist for considerable time and then disap- 
pear, reappearing again after an uncertain period. It has been definitely 
established that, if a patient be syphilitic, a negative Wassermann can be con- 
verted into a positive one by an injection of salvarsan, owing to the fact that the 
spirochete are killed and their toxic action is temporarily increased, the bodies 
concerned in the Wassermann test being thereby increased. This is the so- 
called "Provocative Wassermann Reaction" and can be obtained in every stage 
of infection. The blood for the test should be taken before the injection as a 
control and then 24 and 48 hours after. Occasionally it is wise to take the blood 
one to two weeks after the injection also, in cases which are of doubtful nature. 

In deciding as to the proper criterion of a cure in any case a negative test 
following a positive one has little value. To establish a basis for the assertion 
of a cure, one should insist that the following points be met. One year without 
treatment, without any suspicious signs, with several negative Wassermann 
reactions and no positive ones, and with a negative provocative Wassermann 
reaction and luetin test at the end of the year. x 

/?. Diseases Other Than Syphilis. 2 

The complement-fixation test is, theoretically, applicable to any infectious 
disease. Acute infections, however, do not show positive results until suffi- 
cient time has elapsed to permit of absorption of enough toxin to cause a sys- 
temic response to invasion, as shown by the presence of specific antibodies in 
the serum tested. Sub-acute and chronic cases offer the best chances for posi- 
tive results. Practically, however, the tests have not been equally satisfactory 
in all types of infection or invariably reliable in the same type. 

The principle of the test is the same as described above no matter what 
the condition under investigation. The hemolytic system may be either sheep 
or human, the reagents used being as mentioned with the exception of the anti- 
gen employed. These antigens are polyvalent in character, being prepared 
from several different strains of the suspected organism. As different strains 

1 See Nichols, Jour. Am. Med. Assn., 1912, LVIII, 603; also, Craig and Nichols, Studies 
of Syphilis, Bull. 3> War Dept., Office of Sur. Gen., Washington, 1913; Pusey, Am. Jour. 
Med. Sc, 1913, CXLVI, 497. Corbus (New York Med. Jour., 1914, C, 472) reports a case of 
second infection, the Wassermann having been negative for one and one-half years. 

2 See Ludke in Handbook of Kraus and Levaditi, Erganzungsband 1, ion, 518; 
Ztschr. f. klin. Med, 1911, LXXII, 545. & & 



THE BLOOD. 637 

of the same organism often differ markedly one from another, the possibility 
exists of the serum of an infected patient fixing complement only in the presence 
of closely allied strains. Hence, the more strains of the organism in any given 
antigen the more certain will be the results of the test. 

The antigens are prepared as follows: Cultivate as many different strains 
as possible of the organism on appropriate media, such as agar slants or blood- 
agar slants, and wash the surface of each slant with 2 or 3 c.c. of a 0.9 per cent, 
solution of sodium chlorid, scraping the organisms from the media with a plat- 
inum loop. Pour off the suspension into a sterile tube and shake thoroughly. 
Kill the organisms by heating the tube in a water-bath at 6o° C. for one-half 
hour. Standardize the number of bacteria so that 1 c.c. of the suspension con- 
tains 1,000 million bacteria (see last chapter). The amount of antigen to be 
used in the tests must then be determined by titration just as described for the 
Wassermann test. 

Gonorrhea. 

In systemic gonorrheal infections the complement-fixation test is becoming 
of considerable importance. The technic is carried out as given above. 
The antigen should consist of at least twelve strains of gonococci. In the 
writer's laboratory the human hemolytic system is employed exclusively and the 
results have been constant and satisfactory. Others prefer the sheep system. 
The same precautions must be followed here as in the Wassermann test, every 
point in the technic being carefully controlled. 

A positive reaction indicates a focus of active gonorrheal infection in the 
system; while a negative result does not exclude this condition, owing to the 
variability in the antigens mentioned above. The influence of treatment 
upon this test is not at all clear, but it must be remembered that all cases treated 
with vaccines will show strong positive reactions. Just how long after such 
treatment is discontinued one may expect to obtain a positive result is still 
unsettled, so that the absolute determination of a cure must rest largely on the 
clinical findings. This is an important point, especially where marriage is 
contemplated, as the possibility of infecting another must be kept in mind. 
Non-infectivity cannot be predicated on a single negative fixation test. 1 

Attempts to use this method, as a diagnostic procedure, have been made 
in typhoid fever, tuberculosis, meningococcic infections, cholera, pertussis, 
small-pox, infection with pyogenic cocci and other infections but the results 
have not been conclusive or reliable. Likewise it has been employed in the 
diagnosis of infection with various animal parasites, such as in echinococcus 

1 See Schwartz and McNeil, Am. Jour. Med. Sc, 1911, CXLI, 693; Swinburne, Arch. 
Diagnosis, 1911, IV, 227; Schwartz, Am. Jour. Med. Sc, 1912, CXLIV, 369; Schwartz and 
McNeil, Ibid., 815; Lenartowicz, Tygodnik lekarski, 1912, VII, 368; Dermatol. Wchnschr., 

1912, LV, 1179; Lespinasse and Wolff, Illinois Med. Jour. ; 1913, XXIII, 26; Smith, Am. Jour. 
Dis. Child., 1913, V, 313; Finkelstein and Gerschun, Berl. kiin. Wchnschr., 1913, L, 1817; 
McNeill, Arch. Pediat., 1913, XXX, 657; McDonagh and Kline, Jour. Path, and Bacteriol., 

1913, XVII, 559; Thomas and Ivy, Arch. Int. Med., 1914, XIII, 143; Smith, Am. Jour. 
Dis. Child., 1914, VII, 230; Williams, Interstate Med. Jour., 1914, XXI, 1198; Kolmer and 
Brown, Jour. Infect. Dis., 1914, XV, 6; Thomas, Ivy and Birdsall, Surg., Gyn. and Obs., 

1914, XXX, 390; Arch. Int. Med., 1915, XV, 265; Irons and Nicoll, Jour. Infect. Dis., 1915, 
XVI, 3°3'> Sormani, Nederl. Tijdschr. v. Geneesk., 1915, 1, 1077; U°l e an d MacKinney, New 
York Med. Jour., 1915, CH, 737. 



638 DIAGNOSTIC METHODS. 

disease; helminthiasis in general and trichinosis. Here, also, the results are 
still too uncertain to warrant the routine use of this test. l 

D. Abderhalden's Sero-diagnosis. 2 

1. Of Pregnancy. 

Abderhalden has recently introduced a biologic test for pregnancy, which, 
he assumes, depends upon the presence of certain ferments found in the serum of 
pregnant animals. This test appears to be fairly certain in its results and should 
prove invaluable to the gynecologist and obstetrician. 

It will be recalled that the injection of foreign protein into the system re- 
sults in the production of certain well-established biologic properties in the 
serum of the animal so injected. Among these properties one finds the forma- 
tion of precipitins, agglutinins, anaphylactogens, etc. Abderhalden has 
shown that the parenteral injection (subcutaneous, intraperitoneal or intra- 
venous) of foreign protein or carbohydrate brings about the appearance in the 
blood serum of proteolytic or amylolytic ferments, as an indication of the effort 
of the system to protect itself against the possible toxic effects of this non-hy- 
drolyzed material. Such protective ferments are, he believes, specific and are 
never found in the serum of normal (non-injected) animals. The blood will 
be seen, therefore, to have acquired definite digestive properties apart from 
that resident to a more or less extent in the leucocytes. A further step, taken 
by Abderhalden and his pupils, has been the demonstration that substances, 
which are native to the system but foreign to the blood, may arise from physio- 
logic or pathologic changes within the system itself and, after absorption into 
the blood, produce quite as definite a response as if the material were introduced 
from without. 

Schmorl and Veit have shown that syncytial cells and portions of the chori- 
onic villi may become detached from the placenta and enter the maternal cir- 
culation, while other placental products (of an unknown nature) presumably 
also are either absorbed or washed into the blood current. As these sub- 
stances are, in part at least, protein in nature, we should have, if the theory 
holds, a production of protective proteolytic ferments in the maternal blood, 
which are capable of digesting placental protein and are more or less specific. 
If, therefore, the serum of a suspected animal be treated with placental protein, 
hydrolytic cleavage must occur with the formation of products capable of more 
or less easy detection, provided the serum contains such ferments. These are 
the principles which form the basis of the test as originated by Abderhalden. 

Since the introduction of this test an enormous amount of work has ap- 
peared dealing with its various phases. The earlier publications seemed to 

1 See Kolmer, Arch. Int. Med., 1912, IX, 220; Hastings, Jour. Am. Med. Assn., 1913, LX, 
1208; Besredka and Manoukhine, Ann. de l'lnst. Pasteur, 1914, XXVIH, 569; Compt. rend. 
Soc. de Biol., 1914, LXXVI, 197; Hastings, Jour. Infect. Dis., 1914, XX, 52 and 72; Garbat, 
Am. Jour. Med. So, 1914, CXLVIII, 84; Friedlander and Wagner, Am. Jour. Dis. Child., 

1914, VIII, 134; Olmstead and Luttinger, Arch. Int. Med., 1915, XVI, 67; Winholt, Jour. 
Infect. Dis. 1915, XVI, 389; Howell, Ibid., 456; Nakajo and Asakura, Ibid., 1915, XVII, 388 
and 400, Kolmer and Strickler, Jour. Am. Med. Assn., 1915, LXIV, 800. 

2 Abderhalden, Abwehrfermente des tierischen Organisms, Berlin, 1914. See, also, 
Vaughan, Jour. Am. Med. Assn., 1914, LXIII, 365; Taylor and Hulton, Jour. Biol. Chem., 

1915, XXII, 59; Adami, Jour. Canadian Med. Assn., 1915, V, 569. 



THE BLOOD. 639 

confirm the finding of Abderhalden that specific ferments were present in 
certain conditions, capable of digesting only so-called homologous protein. 
Many of the studies were adverse, the results being attributed by Abderhalden 
to faulty technic. During the last year numerous researches have been made, 
which seem to strike a somewhat discordant note. At the present time the 
status of this question may be summed up as follows: In every fresh serum, 
male or female, normal or abnormal, ferments are present in the blood which, 
under certain conditions, are capable of causing hydrolytic cleavage of protein. 
Unquestionably, these proteolytic ferments are found in increased amounts in 
cases of pregnancy, carcinoma and other conditions to which the Abderhalden 
test has been applied. These ferments are, however, not to be regarded as 
specific, in the sense that they digest only homologous protein added as a sub- 
strat, as it is very questionable whether they act at all on the tissue used in the 
Abderhalden test. Owing to the presence in the blood of normal antiproteo- 
lytic substances, these normal (and hence not defensive ferments) ferments can- 
not exert their activity. If, however, this so-called "antitrypsin" be removed 
from the blood (1) by extraction with chloroform or ether (Delezenne and Poz- 
erski), (2) by inactivation by addition of 0.2 per cent, acetic acid to the serum 
(Opie), or (3) by adsorption with an organic substrat or substances such as 
kaolin, starch or agar (Plaut, Kjaergaard and others), changes occur in the 
degree of dispersion of the colloidal system and the normal proteolytic ferments 
become free to act. Sera treated in any of the above ways will undergo self- 
digestion and liberate dialyzable products capable of detection by the Abder- 
halden or other methods. The substrat (placental or other tissue) acts exactly 
in this way. Further investigations have shown that this reaction is similar 
in many respects to that of combination of antigen and antibody as previously 
discussed, thus bringing the Abderhalden reaction into the realm of other 
immunity tests. According to Stephan, Hauptmann and, more recently, to 
Bronfenbrenner, the Abderhalden test is possible only when complement is 
present. If complement is missing, as when the serum is heated, any fresh 
serum will activate the test. From the later studies of Bronfenbrenner it seems 
probable that the reaction occurs in two stages: (1) the substrat (antigen) 
combines with the antibody, thus becoming sensitized; (2) this sensitized tissue 
then adsorbs the antitrypsin, leaving the normal proteolytic ferments of the 
blood free to digest the protein of the serum. In other words, the Abderhalden 
test does not depend upon the presence of specific ferments which digest only 
homologous tissue, but upon the presence of specific (probably) antibodies, which 
unite with the substrat and, in so doing, adsorb antitryptic bodies and render 
the normal proteolytic ferments active. In this sense the Abderhalden test is 
to be regarded as specific, as it is certain that positive reactions in suspected 
cases are far more numerous, under the most carefully controlled conditions, 
than are the negative findings. However, the specificity is far removed from 
that claimed by Abderhalden. 1 

1 For discussions of the various points raised in this newer investigation see Gammeltoft, 
Ugeskr. f. Laeger, 1913, LXXV, 1247; Heilner and Petri, Munch, med. Wchnschr., 1913, LX, 
1530; Oeller and Stephan, Ibid., 1914, LXI, 12, 75 and 425; Plaut, Ibid., 238; de Waele, Ibid., 
364; Flatow, Ibid., 468, 608, 1168 and 1500; Stephan, Ibid., 801; Hauptmann, Ibid., 11 67; 



640 DIAGNOSTIC METHODS. 

Two distinct methods of detecting the presence of these antibodies have 
been devised. The first, the optic method, is capable of very wide application 
to the diagnosis of different conditions and should prove extremely useful in 
solving many problems of great clinical importance. It requires, however, 
considerable skill and technical ability as well as rather expensive apparatus. 
The second, the dialyzation method, is much simpler both in technic and in 
necessary equipment. Both of these methods require the most assiduous at- 
tention to the various details given, if any dependence is to be placed upon the 
results of the tests. 

1. The Optic Method. 

The basis of this test is as follows. A solution of placental peptone in 
physiologic salt solution has a definite power of rotating the plane of polarized 
light. Likewise, the serum, both suspected and normal, has a similar action. 
The degree of rotation, however, of either remains permanent for some time 
at 37 C. If a solution of peptone and normal serum be mixed and the degree 
of rotation of this mixture determined, no appreciable change will be observed 
between the initial and final polarimetric readings. If, however, a solution 
of placental peptone, whose polarizing action is known, be treated with a serum 
containing the specific ferments above mentioned, digestion of the peptone 
occurs with the formation of products showing rotatory powers sufficient to 
change the initial rotation of the mixture to quite an extent. These changes 
may be observed at different intervals and interpreted as described later. 

Preparation of Placental Peptone. 

The fresh placenta is made blood-free by cutting it into small pieces and 
placing these under running water for about 15 minutes. Dry the pieces be- 
tween folds of filter paper and place them in about five times their weight of 70 
per cent, sulphuric acid. Allow the mixture to stand for three days at room tem- 
perature and shake the container frequently. At the end of this time, place 
the container in ice-water and dilute the contents with 10 volumes of distilled 
water, stirring constantly. Remove the sulphuric acid by adding approxi- 
mately the calculated amount of finely powdered barium hydrate and complete 
the precipitation with a known solution of this salt, stirring the mixture con- 
stantly. When the reaction of the mixture becomes neutral to litmus paper, 
filter off the barium sulphate. If the filtrate be turbid, refiltration is necessary 

von Domarus and Barsieck, Ibid., 1553; Freund and Brahm, Ibid., 1664; Lindig, Ibid., 1668; 
Beumer, Ibid., 1999; Kjaergaard, Ztschr. f. Immunitats f. Orig., 1914, XXII, 31; de Waele, 
Ibid., 170; Herzfeld, Biochem. Ztschr., 1914, LXIV, 103; Ibid., 1915, LXVIII, 402; Wilhelm 
and Szandicz, Ibid., 1914, LXV, 219; Benech, Compt. rend. Soc. de biol., 1914, LXXVI, 361; 
Bettencourt and Menezes, Ibid., 1914, LXXVII, 162; Michaelis and Lagermarck, Deutsch. 
med. Wchnschr., 1914, XL, 316; Frankel, Ibid., 589; Bisgaard and Korsbjerg, Ibid., 1367; 
Mosbacher and Port, Ibid., 1410; Peiper, Ibid., 1467; Oeller and Stephan, Ibid., 1557; Fried- 
mann and Schonfeld, Berl. klin. Wchnschr., 1914, LI, 348; Lange, Ibid., 785; Csepai, Wien. 
klin. Wchnschr., 1914, XXVII, 804; Franz, Arch, f . Gynak., 1914, CII, 579; Heinemann, Monats- 
schr. f. Geburtsh. u. Gynak., 1914, XXXIX, 768; Adachi, Ztschr. f. Geburtsh. u. Gynak., 
1914, LXXVI, 516; Jobling and Petersen, Jour. Exper. Med., 1914, XIX, 480; Ibid., 1914, XX, 
37; Bronfenbrenner, Ibid., 1915, XXI, 221 and 480; Jobling, Eggstein and Petersen, Ibid., 
239; Jobling, Petersen and Eggstein, Ibid., 1915, XXII, 129 and 141; Echols, Jour. Am. Med. 
Assn., 1914, LXIII, 370; Eggstein, Ibid., 735; Falls, Ibid., 1172; Ibid., 1915, LXIV, 1898; 
Ibid., 1915, LXV, 524; Bronfenbrenner, Schlesinger and Mitchell, Ibid., 1915, LXV, 1268; 
Bronfenbrenner, Jour Lab. and Clin. Med., 191 5, I, 79. 



THE BLOOD. 641 

until a perfectly clear nitrate is obtained. The separation of the barium sul- 
phate is much facilitated by the use of the large centrifuge, if such be at hand. 
Wash the precipitate with a large amount of cold water and combine the fil- 
trate and washings. Test the mixture for both barium and sulphuric acid. 
If either be present, it must be removed. Now evaporate this barium- and 
sulphuric acid-free solution to dryness on the water-bath, under reduced pres- 
sure at a temperature not exceeding 40 or 50 C. It is wise to test the evapo- 
rated material at several intervals for the presence of either barium or sul- 
phuric acid, as these sometimes appear on concentrating the mixture. It is 
important that these be removed, as their presence will result in further hydro- 
lysis of the peptone and, in consequence, will lessen the value of the final prod- 
uct. A thick yellow syrup or a foamy mass remains after this evaporation. 
The product may be used in this form, but it is preferable to purify it, if reliable 
results are to be invariable. 

This yellowish residue is dissolved in methyl alcohol with the aid of heat 
and the hot solution is poured into absolute ethyl alcohol. The peptone is 
thrown down as a yellow powder, which is soluble in water to a clear yellowish 
solution of weakly acid or amphoteric reaction. This powder is not hygro- 
scopic. A further purification is still advisable. Dissolve the above yellow 
powder in water up to a 5 per cent, solution and add 10 per cent, solution of 
phosphotungstic acid as long as a precipitate forms. Filter and wash several 
times with water. Rub up this precipitate in a mortar with some water and 
twice its weight of barium hydrate. Filter again and remove the excess of 
barium from the filtrate with sulphuric acid. Filter off the barium sulphate 
and evaporate the filtrate to dryness under reduced pressure at 40 to 50 , as 
outlined above. This product is snow white and is permanent. 

It is absolutely essential for the successful application of the optic test 
that the placental peptone be as pure as possible. The same product is not 
always obtained by the above method, as the hydrolysis may proceed further 
than the peptone stage. Such products are unsuitable for the test. It is wise, 
therefore, to work as quickly and as carefully as possible with a large amount 
of placental substance, so that one may obtain an appreciable amount of pla- 
cental peptone. If the product be found serviceable, it may be kept for years. 
A further point to be considered in the use of a prepared peptone is that the 
solution of this placental product must give absolutely no turbidity with the 
serum to be tested. Such a finding is not infrequent, owing to the probable 
presence of precipitins in the product. Such a peptone cannot be used. 

A further important property of the prepared peptone must be its power 
of rotating the plane of polarized light. The degree must not be too small or 
the product will prove of little value. It will be seen, therefore, that the prepa- 
ration of a serviceable and proper placental peptone is a matter of considerable 
difficulty and is paramount to the successful performance of the test. 

One may preserve the peptone, prepared as above, either in the solid state 
or in the form of a solution. The advantage of a solution is that one has on 
hand a large amount of material, which will give good comparative results, 
as the solution is permanent. Abderhalden formerly used solutions of 0.5 to 

41 



642 DIAGNOSTIC METHODS. 

2.5 per cent, strength. He now advises a 10 per cent, solution of the placental 
peptone in physiologic salt solution. This solution must be absolutely clear 
and colorless. If not, filter through thick paper or a Berkefeld. Preserve 
this clear solution by overlaying its surface with toluol. When required for the 
test, the solution is withdrawn by a pipet dipping below the toluol. If care be 
taken to keep a layer of toluol over the solution, the stock material will be per- 
manent for a long period. Should this solution become turbid at any time, the 
material should be thrown away and a new stock solution prepared as above. 
The optical activity of this stock solution must be tested before each test. 1 

Obtaining The Serum. 

The serum of the patient is obtained as described under the complement- 
fixation tests, withdrawing 10 to 15 c.c. of blood. Place the blood, as drawn, 
directly into a sterilized centrifuge tube, so that all cellular elements may be 
completely separated. For a successful test the serum must show no sign 
of the presence of cells. A further precaution to be taken is that no sign of 
hemolysis must be present in the serum. For this reason, the cells should be 
separated rapidly. It is wise to make the test on the same day on which the 
blood is taken, although, if the precautions above mentioned are observed, a 
delay of 24 to 48 hours does not materially affect the activity of the serum. 

Technic. 

Having prepared the 10 per cent, solution of placental peptone and having 
proven that it answers all the requirements above mentioned, place 1 c.c. of 
this clear solution (withdrawn by a pipet) in a small, clean, sterile test-tube. 
Add 2 c.c. of the clear suspected serum and shake the tube several times. Ex- 
amine the mixture carefully for any turbidity or precipitation. If any be ob- 
served, the test cannot be carried out. Add sufficient physiologic salt solu- 
tion to the mixture to fill the 1 decimeter polarimetric tube (see below). This 
mixture with salt solution is preferably made in this way, rather than to add 
the salt solution after the peptone solution and serum have been placed in the 
polarizing tube. Any turbidity may be much more easily detected. Pour 
the above mixture into the 1 decimeter tube, whose mantle has been filled with 
water at 37 C. 

Carefully determine the initial rotatory power of the mixture, checking the 
readings several times and controlling them by subsequent ones after 5 or 10 
minutes. No change should be observed in these readings. Place the tube and 
its contents in the incubator at 37 C. and repeat the readings every hour for 
a few periods and then continue every 6 or 8 hours. Do not extend the inves- 
tigation over more than 48 hours. Record all readings and interpret them 
as given below. 

Control tubes must be arranged as follows: (1) the peptone solution alone; 
(2) the suspected serum alone; (3) peptone solution plus normal serum; (4) 
peptone solution plus known positive serum; (5) peptone solution plus inacti- 
vated (heated to 6o° C.) suspected serum. In all of these controls the same 

l The placental peptone may now be obtained on the market from the Hochst-Farbwerke 
Co., New York. 



THE BLOOD. 643 

conditions must be maintained and the same length of polarizing tube must 
be used as in the test itself. If any turbidity occurs in any of the control mix- 
tures or tubes, these must be disregarded in interpreting the test as turbid 
solutions give variable results with the polarimeter. 

It goes without saying that this test requires the very best equipment 
possible. The cheap polariscopes are absolutely useless as they are not deli- 
cate enough to detect the fine variations given. The three-shadow instrument 
of Landolt-Lippich, made by Schmidt and Haensch, is especially to be recom- 
mended. The polarizing tubes used are, preferably, the 1 decimeter tubes 
which are furnished with a mantle which may be filled with water at any 
desired temperature. If different length tubes are used in any of the tests, a 
correction must be made in order that comparative figures may be obtained. 

In performing this test, even to a greater degree than when the instrument 
is used in other work, much depends upon the ability of the worker to detect 
slight variations in the degree of the rotatory powers of the mixtures under 
investigation. The method is easy to learn, but the special sensibility toward 
such changes cannot be taught. Abderhalden cautions any one, who shows 
a working error of as much as 0.04 in his observations, against attempting to 
interpret the test. 

In reporting the result of this test, Abderhalden employs the following 
method. 

Deviations within 0.04 Negative 

Deviations between 0.05 and a.i° Positive (+) 

Deviations between o.n and 0.2 Positive(+ +) 

Deviations over 0.2 degree Positive (+ + + ) 

2. The Dialyzation Method. 

This method is much more simple than the optic method, both as regards 
technic and apparatus. It must not be thought, however, that any less care 
is necessary in carrying out the details of the test. In fact, erroneous results 
are, perhaps, more easily obtained by careless manipulation when this method 
is employed. 

The basis of this method is the conversion of the colloidal non-dialyzable 
placental protein into dialyzable products through the activity of the ferments 
above mentioned. These products are, then, detected by simple color reactions 
in the dialysate. 

Preparation of Placental Albumin. 

Although Abderhalden does not regard the proteolytic ferments of the 
serum in pregnancy as absolutely specific for a given species of animal, he, 
nevertheless, insists that the protein preparations used in either of his methods 
be prepared from the placenta of the same species as that of the animal whose 
serum is to be tested. 

As autolysis proceeds fairly rapidly in placental tissue, the albumin should 
be prepared from placentas which are as fresh as possible. Remove the ex- 
ternal portions of the placenta, such as the membranes, and wipe away as much 
blood as' possible. Cut the material into small pieces and wash for a short 



644 DIAGNOSTIC METHODS. 

time in running water. 1 While this is being done, boil about 2 liters of water 
to which are added 2 drops of glacial acetic acid. Throw the washed bits 
of placenta into this boiling water and boil for 5 to 15 minutes. Pour the 
mixture upon a loose quick-acting filter and boil the pieces again with a second 
portion of acidulated water for 5 to 15 minutes. Pour off this water and test 
with the triketohydrinden hydrate reaction given below. If a positive reaction 
obtains, the placental tissue must be again boiled with acidulated water until a 
negative reaction occurs. The essential points in this process are rapid and 
complete coagulation of the placental albumin and the removal of all soluble 
dialyzable material which may react with the reagent mentioned above. 2 

As soon as a negative result is obtained with the extractive water, pour 
the mixture into a wide-mouth flask, add some chloroform, overlay the fluid 
with toluol and stopper the flask; or place the material in several smaller 
glass jars and overlay with toluol. This placental albumin keeps almost in- 
definitely and may be removed from the containers as desired. It should be 
tested, from time to time, to show that it contains, in itself, nothing which may 
react with the reagents used in the later test. 3 

Obtaining The Serum. 

The serum is obtained by venous puncture as previously described, the 
blood (about 10 c.c.) being drawn directly into a sterilized centrifuge tube. 
The cellular elements are separated as quickly as possible and the serum 
drawn off into a clean sterile tube. It is of especial importance in this test 
that the serum show no sign of hemolysis. As it has been shown that amino- 
acids are present in the blood during digestion and may, therefore, give a posi- 
tive reaction with triketohydrinden hydrate, it is wise to take the blood in the 
morning before breakfast in all cases. If such be not done, a less amount of 
serum must be used in the later test to compensate for this possible error. 

Selecting The Dialyzing Tubes. 

It is evident that this part of the preparation for the test is of extreme im- 
portance. The dialyzing thimbles must be permeable for peptone but not for 
albumin. Unless these conditions obtain, the test is valueless. Not all of the 
thimbles, purchasable upon the market, are by any means available. Abder- 
halden advises the use of the diffusion shells No. 5 79 A. of Schleicher and Schiill. 
Not all of these will answer the purpose. 

It is necessary, therefore, that all the dialyzing thimbles used in the test 
should have been previously tested and known to answer the above require- 
ments. As the thimbles are usually dry and hard when obtained, soak them 
in cold water for a few hours, place them in boiling water for a few seconds 
and keep them in water covered with toluol. 

To test these thimbles for their permeability for albumin, proceed as 
follows. Remove the thimble from the water and place in it 5 c.c. of serum 

1 It is essential that all visible blood be removed as its presence will introduce a large error. 

2 Care must be taken not to add an excess of acetic acid to the water as this may interfere 
with the ninhydrin test and thus give rise to an appreciable error. 

3 Abderhalden recommends that this placental albumin be tested with ninhydrin before 
being used in any test. This is a vital point. 



THE BLOOD. 645 

or of a solution of egg albumin. Add a few drops of toluol to prevent bacterial 
action. Place 20 c.c. of distilled water in the dialyzing vessel and overlay this 
with toluol. This dialyzing vessel should be quite narrow, the distance be- 
tween the wall and the thimble (when in place) being about 1/4 cm. These 
vessels are kept plugged with cotton and are sterilized before use. Now sus- 
pend the thimble with its albuminous contents in the dialyzing tube in such a 
way that the fluid outside is as high or, preferably, a little higher than that within 
the thimble. Plug the vessel with cotton to prevent contamination and put the 
apparatus in the incubator at 37 C. for 18 to 24 hours. At the end of this time, 
test the dialysate (outside fluid) for albumin by the biuret or triketohydrinden 
hydrate reactions given below. Those thimbles giving negative results are 
retained to be tested for their permeability to peptone. The shells permitting 
the passage of albumin cannot be used in the test. 

Select those thimbles showing impermeability to albumin and wash them 
thoroughly in water. Place in them 5. c.c. of a 1 to 1000 solution of Witte's 
or, preferably, peptone from silk (peptone La Roche) and add sufficient toluol 
to cover the solution. Dialyze as above against 20 c.c. of distilled water, 
placing the apparatus in the incubator for 18 hours at 37 C. Those thimbles 
which permit the passage of peptone, as shown by the triketohydrinden hydrate 
test, are then kept for use in these tests and the non-permeable ones are laid 
aside. The properly tested and selected thimbles are then preserved in water 
overlaid with toluol. 

Technic. 

Remove a few pieces of the coagulated placental albumin from the con- 
tainer, wash in distilled water and dry between filter paper. 1 Break this up 
into very small bits or grind up in a mortar. Weigh out three portions of 1/2 
gram each. Place 1/2 gram in each of three tested dialyzing thimbles in such a 
way that none of the material touches or remains upon the top or outside of 
the shells. Carefully wash off the outside of the thimble by means of a stream 
of distilled water or hold it under running water. This is done to remove any 
possible adhering placental albumin, which would vitiate the test later made. 
Now add to tube No. 1, 1 to 1.5 c.c. of clear hemoglobin-free serum to be tested. 
This serum is withdrawn from its container by means of a sterile graduated 
pipet. Overlay the surface of the mixture in the thimble with toluol. This 
thimble is then placed in a sterile dialyzing tube, as described above, containing 
20 c.c. of distilled water, which should stand slightly higher than the fluid in 
the thimble. Overlay the external fluid with toluol and plug the dialyzing 
vessel with cotton to prevent contamination. Place the apparatus in the 
incubator at 37 C. for 18 hours and then test the dialysate for peptone as out- 
lined below. 

Controls. 

Charge thimble 2 with 1/2 gram of placental albumin and 1 c.c. of serum 
of a known positive control. Overlay with toluol and arrange as above. 

1 Test the material before use with the triketohydrinden hydrate reaction. Absolutely 
no trace of a blue coloration should obtain . 



646 DIAGNOSTIC METHODS. 

Charge thimble 3 with 1/2 gram of placental albumin and 1 c.c. of a known 
negative serum or with the inactivated (heated to 6o° C.) serum used in the test. 

A further control should be run, using 1 c.c. of the serum alone without 
the addition of albumin, to prove that it does not contain any dialyzable sub- 
stances which will give the later reactions. 

The tests with all of these controls are carried out exactly as the test it- 
self, every precaution being taken to prevent the introduction of errors. The 
tests for cleavage products of albumin are made with one of the following 
tests, the latter being in some respects preferable. 

The Biuret Test. 

This was the test formerly employed by Abderhalden and has some ad- 
vantages in that it does not react with certain dialyzable products not infre- 
quently present in the serum of normal subjects. It requires considerable 
care and skill in manipulation as well as in interpretation. Doubtful results 
are very frequent unless every precaution be taken. 

Remove about 10 c.c. of the dialysate by means of a pipet dipping below 
the toluol. Place this in a test-tube and add 5 c.c. of a 33 per cent, sodium 
hydrate solution. Mix by careful shaking and add very carefully, drop by 
drop, from a buret a very dilute (0.25 per cent.) solution of copper sulphate in 
such a way that a distinct contact ring is formed. If peptone is present, a 
violet-red to a pure red contact ring will be observed, sharply differ entiated 
from the lower colorless and upper blue solutions. This is a positive reaction. 
A negative result is shown by the appearance of a distinctly blue ring. It is 
not the simplest matter to distinguish between the various shadings which occur, 
so that one must not make his decision without having had some experience in 
differentiating the colorations obtained with pure albumin and peptone solutions. 

The Triketohydrinden Hydrate Reaction. 

This reagent occurs in colorless crystals readily soluble in water. It 
may be obtained from the Hochst-Farbwerke (Lucius & B riming) Company 

under the trade name of "ninhydrin. " Its formula is C 6 H 4 <^ yC(OH) 2 . 

It is of especial importance that every precaution be taken to prevent error 
when this reagent is used, as reactions may rise from the presence of substances 
which are not at all associated with the hydrolytic products of protein material. 
This reagent is not by any means specific, even for albumin, peptone or amino- 
acids, although it was formerly believed that it reacted only with substances 
containing an amino and a carboxyl group, the former especially in the a posi- 
tion. It has been shown that there is a large number of compounds which are not 
in a chemical sense combinations with amino and carboxyl groups and which, 
nevertheless, give very characteristic reactions. Among these we find: amines; 
amino-aldehydes; urea derivatives; amino-sulphonic acids; ammonium deriva- 
tives of certain organic acids, dicarbonyl compounds and halogen-aldehydes; 
ammonium compounds of thiosulphuric, oxy-sulpharsenic and selenic acids; 
ammonium formate, ammonium thio-lactate, etc. Of special importance is the 
fact that a very small amount of basic products of putrefactive origin will give a 



THE BLOOD. 647 

decided reaction (hence the importance of using every means to prevent de- 
composition of the tissue). 

Certain further precautions are essential. If any of the original placental 
albumin be left on the outside of the dialyzing thimble, a very obvious error will 
arise. If the serum used contains amino-acids, the amount must be determined 
by depth of color with the ninhydrin. One must avoid the presence of acid or 
ammoniacal fumes in the laboratory. Strong alkalies cause, in themselves, a 
coloration with the reagent, while dilute alkalies may decolorize the solution. 
Acids prevent the appearance of the blue color and will destroy the color already 
formed, even in the presence of a large amount of reacting material. It must 
be insisted, therefore, that the fluid to b.e tested be absolutely neutral. Further, 
all vessels and pipets must be absolutely clean and the water used must be free 
from bacteria. 1 

Remove 10 c.c. of the dialysate by means of a pipet dipping below the 
toluol and place this in a large test-tube. Add 0.2 c.c. of a 1 per cent, aqueous 
solution of triketohydrinden hydrate. Heat rapidly to the boiling point and 
keep the mixture boiling for one minute. If the reaction be negative, the 
solution remains colorless or becomes, at most, light yellow. If the reaction 
be positive, a deep blue color will appear either immediately or on allowing 
the tube to stand for a short time. After use wash the thimbles thoroughly in 
running water and then place them in boiling water for not over 15 seconds. 

The reaction is carried out in the same way with the control tubes. Tube 
2 should show a distinct positive reaction, while tube 3 should give a negative 
result. The tube with serum alone should show a negative reaction, but oc- 
casionally it is positive owing to the presence of a large amount of ammo-acids 
in the serum as drawn. If the controls are all positive, the test is, of course, 
valueless, as some factor has been imperfectly controlled. 

Bronfenbrenner's Modification. 

Using the known property of antibodies of uniting with antigen at low tem- 
peratures, which prevent the activity of the complement and, also, exclude the 
acitivity of the proteolytic ferment, Bronfenbrenner adds to 1/ 2 gram of placental 
tissue, contained in a glass tube, 1.5 c.c. of the suspected serum. Place this in 
the ice box over night. The next morning the serum is poured off, the tissue is 
placed in a centrifuge tube and washed, by centrifugation and decantation, until 
all traces of the serum are removed. The sensitized tissue is then placed in a 
dialyzing thimble and 1.5 c.c. of fresh male guinea-pig serum or normal human 
serum are added. Follow the usual technic of Abderhalden from this point. A 
positive ninhydrin reaction, under these conditions, is almost absolute evidence 
of specific disease. 

If the suspected serum, which has been in contact with the placental tissue 

1 See Ruhemann, Jour. Chem. Soc, Trans., 1910, XCVII, 1438 and 2025; Ibid., 1911, 
XCLX, 792; Ibid., 1912, CI, 780; Abderhalden and Schmidt, Ztschr. f. physiol. Chem., 
1911, LXII, 37; Ibid., 1913, LXXXV, 143; Pearce, Jour. Am. Med. Assn., 1913, LXI, 1456; 
Warfield, Ibid., 1914, LXII, 436; Halle, Lowenstein, and Pribram, Biochem. Ztschr., 1913, 
LV, 357; Neuberg, Ibid., 1913, LVI, 500; Herzfeld, Ibid., 1914, LIX, 249; Neuberg, Ibid., 1914, 
LXVTI, 56; Deetjen and Frankel, Munch, med. Wchnschr., 1914, LXI, 466; Howe Biochem. 
Bull., 1914, III, 269. 



648 DIAGNOSTIC METHODS. 

in the ice box, be placed in a dialyzing thimble and kept at 37 C. for 18 hours, 
the dialysate will show a positive reaction due to the removal of the antitrypsin 
and the later self-digestion of the serum protein. The untreated serum, used as 
a control, should show no ninhydrin reaction. 

Value of The Test. 

This test, applied by either one of the above methods, is extremely valu- 
able. Abderhalden's results, which have been definitely confirmed (see bibli- 
ography at the end of this discussion), show that in the majority of cases in which 
the reaction was positive the patient has been proven to be pregnant; while nega- 
tive reactions have been given only in non-pregnant cases. In other words, 
a negative result is definite, a point of distinction from a negative result with 
the complement-fixation test; while a positive result is evidence of the presence 
of specific elements within the blood (pregnancy), either at the time of the test 
or at least within a period which does not exceed two weeks (Schwarz). In the 
absence of a true pregnancy, hydatidiform mole and other pathologic conditions 
of the chorionic villi may be accountable for a positive reaction. A negative 
test should be considered as eliminating pregnancy. 

As this reaction is evidence of pregnancy, in most cases giving positive 
results (an error of 10 per cent, being probable), it is especially interesting to find 
that it is positive from the middle of the second month of pregnancy on and 
disappears within 10 to 15 days after the termination of the pregnancy. It is, 
therefore, of great value in early diagnosis as well as in the diagnosis of a second 
pregnancy following so closely upon a previous one that other signs are not pres- 
ent. Differential diagnosis should be greatly improved by the use of this 
test and fewer laparotomies performed under the mistaken diagnosis of tumor. 
A further value of the test is found in the fact that medico-legal cases may be 
established on a firm basis of definite knowledge of the conditions present. 1 

1 As this test is of so much importance I give a fairly full bibliography of the subject for 
reference. Abderhalden, Freund and Pincussohn, Prak. Ergeb. d. Geburtsh. u. Gynak., 1910, 
II, 367; Abderhalden, Handb. d. biochem. Arbeitsmeth., 1911, V, 575; Ibid., 1912, VI, 223; 
Abderhalden and Kiutsi, Ztschr. f. physiol. Chem., 1912, LXXVII, 249; Abderhalden, Ibid., 
1912, LXXXI, 90; Ibid., 1912, LXXXII, 109; Munch, med. Wchnschr., 1912, LIX, 1190, 
1305, 1939, and 2172; Deutsch. med. Wchnschr., 1912, XXXVIII, 2160; Abderhalden and Weil, 
berl. Tierarztl. Wchnschr., 1912, XXVIII, 665; Frank and Heimann, Berl. klin. Wchnschr., 

1912, XLIX, 1706; Franz and Jarisch, Wien. klin. Wchnschr., 1912, XXV, 1441; Veit, Ztschr. 
f. Geburtsh. u. Gynak., 1912, LXXII, 463; Petri, Zentralbl. f. Gynak., 1913, XXXVII, 235; 
Schwarz, Interstate Med. Jour., 1913, XX, 195; Henkel, Arch. f. Gynak., 1913, XCIX, 56; 
Lindig, Munch, med. Wchnschr., 1913, LX, 288; Abderhalden, Ibid., 411 and 462; Jellinghaus 
and Losee, Bull. Lying-in Hosp., New York, 1912, IX, 68; Gutman and Druskin, Med. Record, 

1913, LXXXIV, 99; Engelhorn, Munch, med. Wchnschr., 1913, LX, 587; Schlimpert and 
Hendry, Ibid., 681; Freund and Brahm, Ibid., 685; Freund, Ibid., 700 and 763; Abderhalden, 
Ibid., 701 and 763; Lindig, Ibid., 702; Heimann, Ibid., 915; Stange, Ibid., 1084; Riibsamen, 
Ibid., 1139; Schiff, Ibid., 1197; Kin g> Ibid., 1198; Maccabruni, Ibid., 1259; Ann. diostet., 
Milano, 1913, I, 486; Abderhalden, Munch, med. Wchnschr., 1913, LX, 1386; Lampe and 
Papazolu, Ibid., 1423 and 1533; Frank and Rosenthal, Ibid., 1425; Lichtenstein, Ibid., 
1427; Heilner and Petri, Ibid., 1530 and 1775; Steising, Ibid., 1535; Frank, Rosenthal and 
Biberstein, Ibid., 1594; Abderhalden and Weil, Ibid., 1703; Schlimpert and Issel, Ibid., 1758; 
Bruck, Ibid., 1775; Goudsmit, Ibid., 1775; Abderhalden and Schiff, Ibid., 1923; Plotkin, Ibid., 
1942; Lampe andFuchs, Ibid., 2112; Tschudnowsky, Ibid., 2282; Behne, Zentralbl. f. Gynak., 
1913, XXXVII, 613; Parsamow, Ibid., 934; Mayer, Ibid., 1181; Porchownick, Ibid., 1226; 
Schlimpert, Berl. klin. Wchnschr., 1913, L, 1136; Rosenthal, Ibid., 1149; Gottschalk, Ibid., 
1151; Veit, Ibid., 1241; Aschner, Ibid., 1243; Schafer, Ibid., 1605; Evler, Ibid., 1606; Ebeler 
and Lohnberg, Ibid., 1898; Williams and Pearce, Surg., Gynec. and Obst., 1913, XVI, 411; 
McCord, Ibid., 418; Judd, Jour. Am. Med. Assn., 1913, LX, 1947; Am. Jour. Med. So, 1913, 



THE BLOOD. 649 

2. Of Other Conditions. 

The principles upon which Abderhalden's sero-diagnosis is supposedly 
based have been considerably altered by the newer researches. However, the 
method has found, and will continue to find, wide application in the diagnosis of 
varied conditions. 1 Although the basic idea of the presence of specific ferments 

CXLVI, 391; Pari, Gazz. d. osp., 1913, XXXIV, 727; Ekler, Wien. klin. Wchnschr., 1913, 
XXVI, 696; Jaworski and Szymanowski, Ibid., 922; Daunay and Ecalle, Compt. Rendu Soc. 
de biol., 1913, LXXrV, 1190; Sunde, Norsk Mag. f . Laegevid., 1913, LXXIY, 1234; Schlimpert, 
Brit. Med. Jour., 1913, II, 1003; Deutsch. med. Wchnschr., 1913, XXXIX, 1225; Jonas, Ibid., 
1099; Naumann, Ibid., 2086; Evler, Med. Klin., 1913, IX, 1042 and 1086; Bauer, Ibid., 1797; 
Abderhalden, Monatschr., f. Geburtsh. u. Gynak., 1913, XXXVIII, 24; Wolff, Ibid., 394; 
Schwarz, Interstate Med. Jour., 1913, XX, 393; Jour. Am. Med. Assn., 1913, LXI, 484; Abder- 
halden, Gynak. Rundschau, 1913, VII, 467; Decio, Ibid., 436; Ann. di ostet, 1913, I, 198; 
Abderhalden and Fodor, Ztschr. f. physiol. Chem.,'i9i3, LXXXVII, 220; Abderhalden and 
Schiff, Ibid., 225 and 231; Gabastou and Widakowich, Semana Med., 1913, XX, 813; Scherer, 
Berl. klin. Wchnschr., 1913, L, 2183; Williamson, Jour. Obst. and Gynec. Brit. Emp., 1913, 
XXIV, 211; Werner and von Winiwarter, Wien. klin. Wchnschr., i9i3,XXVI, 1841; Labus- 
quiere, Ann. de Gynec. et d'Obst, 1913, XL, 664; Fraenkel, Berl. klin. Wchnschr., 1913, 
L, 2287; Kabanoff, Med. Obozr., 1913, LXXX, 312; Sabin, Presse med., 1913, XXI, 1016; 
Williams and Ingraham, Colorado Med., 1913, X, 364; Abderhalden, Munch, med. 
Wchnschr., 1913, LX, 2774; Lampe, Ibid., 2831; Meyer, Ibid., 2906; Wegener, Ibid., 1914, 
LXI, 15; Bronstein, Ibid., 74; Oeller, Stephan and Mayer, Ibid., 12 and 75; Behne, Zen- 
tralbl. f. Gynak., 1914, XXXVIII, 74; Akimoto, Ibid., 81; Diner, New York Med. Jour., 

1914, XCLX, 478; Holmes, Chicago Med. Recorder, 1914, XXXVT, 213, 475 and 593; Schwarz, 
Jour. Am. Med. Assn., 1914, LXIII, 371; Lindemann, Ztschr. f. d. ges. exper. Med., 1914, IV, 
177; Guggenheimer, Ztschr. f. Chemotherap., 1914, III (2TI) 210; McLester, Am. Jour. Med. 
Sc, 1914, CXLVIII, 75; Pearce and Williams, Jour. Infect. Dis., 1914, XTV. 351; Pregl, 
Fermentforsch., 1914, I, 1; de Crinis, Ibid., 13; Abderhalden, Ibid., 20; Hirsch, Ibid., ^$; 
Strauss, Ibid., 55; Paquin, Ibid., 58; Ross and Singer, Arch. Int. Med., 19 14, XTV, 552; Ibid., 

1915, XV 724; Lange, Biochem. Ztschr., 1914, LXI, 193; Thar and Kotschnieff, Ibid., 1914, 
LXIII, 483; Salus, Ibid., 1914, LXV, 381; Parsamow, Ibid., 1914, LXVI, 269; Neuberg, Ibid., 
1914, LXVII, 56; Halsen, Ibid., 277; Frankel, Ibid., 298; Thar and Kotschnieff, Ibid., 1915, 
LXLX, 389; Abderhalden, Deutsch. med. Wchnschr., 1914, XL, 268 and 401; Hirsch, Ibid., 
270; Allmann, Ibid., 271; Pincussohn, Ibid., 425; Abderhalden, Ibid., 428; Lampe and Stroo- 
mann, Ibid., 635; Lampe, Ibid., 1213; Melikjanz, Ibid., 1369; Nieszytka, Ibid., 1519; Hirsch. 
Ibid., 1560; Otto and Blumenthal, Ibid., 1836; Eder, Ibid., 1838; Abderhalden, Munch, 
med. Wchnschr., 1914, LXI, 233 and 401; Voelkel, Ibid., 349; Singer, Ibid., 350; Lampe, Ibid., 
463; Kammerer, Clausz and Dieterich, Ibid., 469; Swart and Terwen, Ibid., 603; Abderhalden 
and Grigorescu, Ibid., 767; Abderhalden and Fodor, Ibid., 765; Schiff, Ibid., 768; Abderhalden, 
Holle and Strauss, Ibid., 804; Abderhalden and Paquin, Ibid., 806; Deetjen and Frankel, Ibid., 
826; Abderhalden and Wildermuth, Ibid., 862; Rosenthal and Biberstein, Ibid., 864; Abder- 
halden and Ewald, Ibid., 913; Lichtenstein and Hage, Ibid., 915; Freymuth, Ibid., 916; 
Griesbach, Ibid., 979; Abderhalden and Grigorescu, Ibid., 1209; Herzfeld, Ibid., 1503; Abder- 
halden, Ibid., 1897; Jaffe and Pribram, Ibid., 2125; Nieden, Ibid., 2200; Jaffe and Pribram, 
Ibid., 1915, LXII, 614; Puppel, Monatsschr. f. Geburtsh. u. Gynak., 1914, XXXIX, 764; Fetzer, 
Ibid., 1 914, XL, 598; Petri, Ibid., XLI, 309 and 388; Baumann, Ibid., 191 5, XLII, 
199; Wallis, Jour. Obs. and Gynec. of Brit. Emp., 1914, XXV, 53; Paine, Boston Med. and 
Surg. Jour., 1914, CLXX, 303; Ecalle, Arch. mens. d'Obs. et de Gynec, 1914, III, 257; Partos 
and d'Ernst, Ibid., S33\ Jellinghaus and Losee, Am. Jour. Obs. and Dis. Women and Child., 
1914, LXLX, 593; Kolmer and Williams, Ibid., 1915, LXXI, 899; Ibid., 1915, LXXII, 101; 
Grey, Bull. Johns Hopkins Hosp., 1914, XXV, 117; Echols, Wis. Med. Jour., 1914, XIII, 1; 
Stoner and Skeel, Cleveland Med. Jour., 1914, XIII, 392 ; Bullock, Lancet, II, 225; Gavronsky, 
Ibid., 1915, I, 119; Leitch, Brit. Med. Jour., 1914, II, 161 and 330; Hinselmann, Zentralbl. f. 
Gynak., 1914, XXXVIH, 258; Kjaergaard, Ibid., 264; Schottlaender, Ibid., 425; Primsar, Ibid., 
438; Hussy, Ibid., 897; Gentili, Ibid., 1159; Saxl, Berl. klin. Wchnschr., 1914, LI, 824; Brieger 
and Schwalm, Ibid., 839; Ebeler and Lohnberg, Ibid., 1915, LII, 319; Abderhalden, Med. 
Klin., 1914, X, 665; Lampe and Paregger, Ibid., 725; Abderhalden and Grigorescu, Ibid., 728; 
Forster, Ibid., 772; von Graff and Saxl, Ibid., 1387; Lindstedt, Hygiea, 1915, LXXVII, 833; 
Goldstone, Med. Rec, 1914, LXXXVI, 67; Keitler and Lindner, Wien. klin. Wchnschr., 1915, 
XXVIII, 549; Goodman, Ann. Surg., 191 5, LXI, 149. 

1 See Wolff and Frank, Berl. klin. Wchnschr., 1914, LI, 875; Gumpertz, Beitr. z. Klin, 
der Tuberk., 1914, XXX, 201; Gwerder and Melikjanz, Munch, med. Wchnschr., 1914, LXI, 
980; Ammenhauser, Ibid., 2000; Schultz and Grote, Ibid., 2510; Baeslack, Jour. Am. Med. 
Assn., 1914, LXII, 1002; Ibid., 1914, LXIII, 559; Luxenburg, Med. Klin., 1914, X, 1104; 
Varney and Morse, Michigan State Med. Jour., 1914, XIII, 515; Hegner, Cor.-Bl. f. schweiz. 



650 DIAGNOSTIC METHODS. 

in the blood in pathologic conditions must, probably, be changed, yet the test 
appears to be somewhat specific in the sense that certain antibodies are probably 
present in the serum, which antibodies so sensitize the substrat that theanti- 
proteolytic substance of the blood is withdrawn, thus leaving the protein of the 
serum free to undergo self -digestion by the normal proteolytic ferments of the 
blood. The possibility unquestionably exists that the use of a definite substrat 
(homologous in nature) may enable us to detect and differentiate pathologic 
processes much more sharply than has hitherto been the case. Although the 
specific nature of this test, as originally advocated by Abderhalden, has been 
attacked and shown to be dependent on other factors than those originally 
assumed, yet it is certain that there is much diagnostic value to the test, whatever 
may be its explanation. Considerable study must, however, be made before we 
are in a position properly to interpret our findings in all cases. 

(a) Cancer. 

In no other condition is a certain and constant diagnostic test so much to 
be desired as in cancer. Many methods have been advocated but Abderhalden's 
test is by far the most promising. 

The technic of the test is as described above. As the protein material to 
be sensitized, cancerous tissue is employed, the material being cut into small 
bits and prepared exactly as is placental tissue. It would seem necessary to 
use homologous tissue in this work, that is carcinomatous or sarcomatous as the 
case may be. This point has not been definitely established but the results in 
any given case would, probably, be more specific. The dialyzation fluid is 
distilled water. Erpicum advises the use of a 2 per cent, solution of sodium 
rluorid as an antiseptic, but this is unnecessary if toluol is used freely as should 
be done. Sterility of all material and glass-ware is essential in this test. 

The results reported have been more than encouraging in that benign 
growths seem to give, almost constantly, negative results while the malignant 
forms show an extremely large percentage of positive findings. The writer 
has used this test frequently and has corroborated his findings by pathological 
examination of the tissues removed at operation in many of the cases. In some 
of the cases the results have been far from satisfactory. 1 More detailed study 
is necessary before the value of this test in cancer is determined. 

Aerzte, 1914, XLIV, 1292; Vladesco and Popesco, Compt. rend. Soc. de. biol., 1914, LXXYII, 
586; Smith, Jour. Infect. Dis., 1915, XVI, 319; Falls, Ibid., 466; Yoelkel, Munch, med. Wchn- 
schr., 1 9 14, LXI, 349; Wohl, Am. Jour. Med. Sc, 191 5, CXLIX, 427. 

1 See Abderhalden, Munch, med. Wchnschr., 19 13, LX, 2385; Labbe, Gaz. med. de Nantes, 
1913, 2 me. S., XXXI, 461; Frank and Heimann, Berl. klin. Wchnschr., 1913, L, 631; Markus, 
Ibid., 776; Munzer, Ibid., 777; Epstein, Wien. klin. Wchnschr., 1913, XXVI, 649; Deutsch 
and Kohler, Ibid., 1361; Erpicum, Bull, de l'Acad. roy. de med. de Belg., 1913, XXVII, 624; 
Ludke, Gaz. d. hop., Paris, 1913, LXXXVI, 1064; von Gambaroff, Munch, med. Wchnschr., 
1913, LX, 1644; Brockman, Lancet, 1913, II, 1385; Abderhalden, Deutsch. med. Wchnschr., 

1913, XXXIX, 2391; Hara, Ibid., 2559; Fried, Munch, med. Wchnschr., 1913, LX, 2782; 
Schwarz, Am. Jour. Obst, 1914, LXIX, 54; Ball, New York Med. Jour., 19 13, XCVIII, 
1249; Jour. Am. Med. Assn., 1914, LXII, 599; Halpern, Mitt. a. d. Grenzgeb. d. Med. u. Chir., 

1914, XXVII, 340; Piorkowski, Berl. klin. Wchnschr., 1914, LI, 254; Frankel, Ibid., 356; 
Fasiam, Wien. klin. Wchnschr., 1914, XXVII, 267; Manoiloff, Ibid., 269; Oeller and Stephan, 
Munch, med. Wchnschr., 1914, LXI, 579 and 583; Schawlow, Ibid., 1386; Weinberg, Ibid.- 
1617, 1685 and 1732; Frankel, Deutsch. med. Wchnschr., 1914, XL, 589; Hara, Ibid., 1258; 
Cytronberg, Mitt. a. d. Grenzgeb. der Med. u. Chir., 1914, XXVIII, 243; Heimann and Fritsch, 



THE BLOOD. 651 

(b) Dementia Praecox. 

In the realm of nervous disorders differential -diagnosis is not always as 
exact as could be desired. Abderhalden's test has been applied by many in such 
cases with excellent results. In this work the selection of the proper material 
for the substrat seems to be the matter of greatest importance. It is probable 
that testicular tissue should be used for male patients and ovarian tissue for 
female sufferers, although cortical tissue and thyroid have been used by some 
but positive results with the latter are few. The adoption of cortical tissue is 
highly improbable as differential diagnosis is not subserved by its use. General 
paralysis, epilepsy with dementia, and manic-depressive insanity all respond 
promptly to brain tissue but very rarely to testicular or ovarian material. A 
further point is that all syphilitic and parasyphilitic disorders seem to cause 
hydrolysis of cortical brain tissue but not of other organs. The antibodies 
found in the serum of these patients are, therefore, more or less specific, as a 
reaction with testicular or ovarian tissue is almost positive evidence of dementia 
praecox. 1 

E. Herman-Perutz Reaction. 

As the Wassermann test for syphilis is so complex and requires so much 
experience for its proper performance and interpretation, attempts have been 
made to find a serum reaction for syphilis which could be used by the general 
worker and which, at the same time, would give results comparable to those of 
the Wassermann test. Such a test is that of Herman and Perutz, 2 which is a 



Arch. f. Chir., 1914, CIII, 659; Lanzarini, Gazz. d. osp., 1914, XXXV, 1057; Trubina, Deutsch. 
Zeitsch. f. Chir., 1914, CXXXI, 520; Fulchiero, Rif. med., 1914, XXX, 1261; Van Slyke and 
Vinograd, Proc. Soc. Exper. Biol, and Med., 1914, XI, 154; Dick, Jour. Infect. Dis., 1914, 
XIV, 242; Kahn, Arch. Diag., 1914, VII, 356; Biehn, 111. Med. Jour., 1915, XXVII, 206; 
Goodman, Surg., Gyn. and Obs,, 1914, XIX, 797; Levin, New York Med. Jour., 1914, C, 621; 
Bullock, Lancet, 1915, I, 223; Goodman and Berkowitz, Surg., Gyn. and Obs., 1915, XXI, 
463; Ball, Jour. Am. Med. Assn., 1914, LXII, 599; Ibid., 1914, LXIII, 1169; Lowy, Ibid., 
1915, LXTV, 1559; Levin and Van Slyke, Ibid., 1915, LXV, 945; Berghausen, Interstate Med. 
Jour., 1915, XXII, 228. 

1 See Fauser, Deutsch. med. Wchnschr., 1913, XXXLX, 304; Wegener, Munch, med. 
Wchnschr., 1913, LX, 1197; Schulz, Deutsch. med. Wchnschr., 1913, 39, 1399; Lampe, Ibid., 
1774; Bundschuh and Roemer, Ibid., 2029; Fischer, Ibid., 2138; Fuchs, Munch, med. Wchn- 
schr., 1913, LX, 2230; Binswanger, Ibid., 2321; Theobald, Med. Klin., 1913, IX, 1850; Berl. 
klin. Wchnschr., 1913, L, 2180; Beyer, Munch, med. Wchnschr., 1913, LX, 2450; Kafka, 
Med. Klin., 1914, X, 153; Fauser, Munch, med. Wchnschr., 1914, LXI, 126; Kastan, Deutsch. 
med. Wchnschr., 1914, XL, 319; Arch. f. Psychiat, 1914, LIV, 928; Grigorescu, Med. Klin., 

1914, X, 418; Neue, Ibid., 1217 and 1259; Simon, Jour. Am. Med. Assn., 1914, LXII, 1701; 
Sterne, 111. Med. Jour., 1914, XXVI, 327; Gehrmann, Ibid., 335; Holmes, Ibid., 332; New 
York Med. Jour., 1914, XCIX, 567; Chicago Med. Recorder, 1914, XXXVI, 644; Ibid., 

1915, XXXVII, 88; Bouman and Hasselt, Nederl. Tijdschr. v. Geneesk., 1915, I, 423; 
Mayer, Munch, med. Wchnschr., 1915, LXII, 580; Sioli, Arch. f. Psychiat. u. Nervenkr., 1914, 
LV, 241. 

2 Med. Klin., 1911, VII, 60. Landau (Wien klin. Wchnschr., 1 913, XXVI, 1702; see, 
also, Presse med., 1914, XXII, 335, and Riv. osped., 1914, IV, 641) has introduced a test for 
syphilis, using as his reagent a 1 per cent, solution of iodin in carbon tetrachlorid. To 0.2 c.c. 
of fresh serum he adds 0.1 c.c. of the above reagent, shakes the tube and allows the mixture 
to stand for four hours. Positive results are shown by a clear yellow serum above the 
decolorized reagent, indicating the binding of the iodin probably by the lipoids. If the serum 
is an opaque grayish white, the reaction is negative. While this test gives results in many 
cases which parallel those of the Wassermann test, yet it shows a large percentage of positive 
results in non-syphilitic cases. Further, a negative Landau test is of little value as many 



652 



DIAGNOSTIC METHODS. 



modification of an older test of Porges. Its simplicity should recommend it, 
especially as its results are usually definite and easily interpreted. 
For this test two solutions are used. 

Solution A. 

Sodium glycocholate, 2 . 00 grams 

Cholesterin, 0.40 gram 

95 per cent, alcohol, 100.00 c.c. 

At the time of making the test, this solution is diluted with distilled water 
in proportion of 1 to 20. 

Solution B. 

A 2 per cent, aqueous solution of sodium glycocholate. 

This solution must be made up fresh at the time of making the test, as it 
is not permanent. Further, the solution is not a perfect one so that the bottle 
should be shaken before using. 

Technic. 

Withdraw the blood as for the Wassermann test. As only a small amount 
is needed, puncture of the ear or finger is usually sufficient. Collect the blood 
in a sterile tube, allow it to clot and centrifuge. The serum must show no 
hemolysis. Pipet off the clear serum. Although Herman and Perutz believed 
the test was better if the serum was inactivated by heating to 56 C. for one- 
half hour, later work has shown that this is not essential. 

To 0.4 c.c. of the clear serum add 0.2 c.c. of the 1 to 20 dilution of solution A 
and 0.2 c.c. of solution B. Shake thoroughly, plug the tubes with cotton and 
allow them to stand not longer than 18 to 24 hours at room temperature. On 
examining the tubes, a positive result is indicated by a flocculent precipitate, 
while a negative result is shown by no precipitation. The results may be 
expressed as +,+ + ,+ + + depending on the degree of precipitation. This 
gives the results so that comparison with the Wassermann may be made easily. 

This test practically parallels the Wassermann test, except that in secondary 
syphilis the results are not quite so uniform with the former. The percentage 
of variation is greater than with the Wassermann test, so that our interpretation 
of the result would be guarded. In other words, a positive result indicates 
syphilis, while a negative result does not exclude syphilis. This reaction should 
find a place in the laboratory of every general worker who does not have facilities 



known syphilitics give negative tests. This test has very little diagnostic value. See Golay, 
Rev. mid. de la Suisse roman., 19 14, XXXIV, 571; Villaret and Pierret, Presse med., 1914, 
XXII, 582; Correa, Brazil-Medico, 1914, XXVIII, 395; Kolmer, Jour. Am. Med. Assn., 1915, 
LXIV, 146 1 and 1966; Stillians, Ibid., 1964; Capello, Gazz. d. osp., 1915, XXXVI, 423; 
Recupero, Ibid., 854. 



THE BLOOD. 653 

for performing the Wassermann test, but should not be relied upon to the ex- 
clusion of the Wassermann test. 1 

F. Tests before Transfusion. 

As transfusion of blood is becoming a matter of more or less frequent 
occurrence, it is advisable to test the blood for isoagglutinins and isohemolynns 
in every possible case. " That donor should be chosen whose blood shows no 
inter-agglutination or hemolysis with the patient's serum and corpuscles. If 
such a donor cannot be obtained, it is safer to use a person whose serum is 
agglutinative toward the patient's cells than one whose cells are agglutinated 
by the patient's serum" (Kolmer). 

Technic. 

Two or three c.c. of blood are obtained from each donor by the usual sero- 
logical method. Place 0.5 c.c. at once in a centrifuge tube containing 5 c.c. of a 
1 per cent, solution of sodium citrate in normal salt. Place the remainder of the 
blood in a small dry test-tube and allow the serum to separate. Obtain 3 or 4 
c.c. of blood from the recipient in the same manner and follow the same pro- 
cedure with this blood as with that of the donors. 

Centrifuge the sodium citrate tubes, pipet off the supernatant fluid and wash 
the cells again with normal salt solution. Repeat if necessary in order to free 
the cells from every trace of serum. After the final washing, add enough normal 
salt solution to the cells to make a total volume of 5 c.c. 

Centrifuge the serum tubes and separate the clear serum. This serum 
should show no trace of hemolysis. The following series of tests should then be 
set up. In this work the blood should not be allowed to stand for a period of 
time before the elements are separated, and the actual tests should be begun 
within 24 hours of the withdrawal of the blood. Use small sterile test-tubes 
about 8 by 1 cm. 

1. Four drops of donor's serum + 1 drop of recipient's red-cell emulsion. 

2. Four drops of recipient's serum + 1 drop of donor's red-cell emulsion. 

3. Control: Four drops of donor's serum + 1 drop of donor's red-cell 
emsulion. 

4. Control: Four drops of recipient's serum + 1 drop of recipient's red- 
cell emulsion. 

5. Control: One drop of donor's red-cell emulsion + 4 drops of normal 
salt solution. 

6. Control: One drop of recipient's red-cell emulsion + 4 drops of nor- 
mal salt solution. 

One c.c. of normal salt solution is added to each tube and the tubes are 
shaken and placed in the incubator for two hours, being inspected about every 
one-half hour. Agglutination in tubes 1 and 2 may be recognized macroscop- 

1 See Gammeltoft, Hospitalstid., 1912, V, 471; Deutsch. med. Wchnschr., 1912, XXXVIII, 
1934; Jensen and Feilberg, Hospitalstid., 1912, V, 493; Feilberg, Ibid., 973; Ipsen and Helweg, 
Ibid., 1341; Thomsen and Boas, Ibid., 1152; Jensen, Ibid., 1516; Pontoppidan, Ugesk. f. 
Laeger, 1912, LXXTV, 1377; Jensen and Feilberg, Berl. klin. Wchnschr., 1912, XLLX, 1086; 
Lade, Deutsch. med. Wchnschr., 1913, XXXIX, 693; Kallos, Ibid., 1885; Leschly and Boas, 
Hospitalstid., 1913, VI, 640; Thomsen and Boas, Ztschr., f. Immunitatsforsch., 1913, XVI, 430; 
Olitsky and Olmstead, Jour. Am. Med. Assn., 19 14, LXII, 293. 



654 DIAGNOSTIC METHODS. 

ically by the clumping of the red cells. Hemolysis is noted as in previous tests. 
If there is any question about hemolysis, let the tubes stand in the ice box over 
night and read them as in the complement- fixation tests. Tubes 3, 4, 5 and 
6 should show no hemolysis or agglutination. 1 

Besides the above tests, it is advisable to test the donors with the Wasser- 
mann test. Further, immediate members of the patient's family offer the least 
risk in transfusion and should be obtained whenever possible. 

IX. Medicolegal Aspects. 

In medicolegal examinations one is frequently called upon to determine the 
identity of various suspected stains. Such examinations may tax the entire re- 
sources of the worker and have, in many cases, led to absolutely negative results. 
It is not sufficient at the present day that the examiner state that a certain stain 
is blood, but he must be prepared to say what kind of blood it is. This latter 
point has been made possible by recent work so that one is fairly sure of lim- 
iting his statement at least to the blood of very closely associated animals. 

(1). Red Cells. 

It is not always a simple matter to determine that a stain on cloth, wood, 
iron, etc., is really due to blood. The color of the stain may be of any hue 
from dull red to a dirty gray, depending upon its exposure to various elemental 
conditions. The action of various substances, such as mortar, brick, or lime 
in any form, strong acids and alkalies, leather, chemicals in wall-paper, starched 
clothing, etc., may so change the blood or its reactivity to certain tests that no 
definite conclusions can be obtained. 

It is self-evident that the demonstration under the microscope of blood- 
cells is the surest proof of the presence of blood, the size and shape of the 
corpuscles frequently giving a clew as to the source of the blood. It becomes, 
therefore, necessary to make a suspension of the stained material preferably in 
isotonic (0.9 per cent.) sodium chlorid solution.. The degree of solubility 
of the stain will depend upon the age of the stain, the heat to which it has 
been subjected, the amount of sunlight or of moisture to which it has been 
exposed, and the material upon which the stain is formed. Various fluids have 
been advanced, from time to time, as dissolving agents for the stains, many of 
these are active laking or hemolytic agents, especially for dried red cells, 
so that one sees in such preparations only the shadow of the red cell. Such 
laking agents are distilled water, 0.85 per cent, ammonium chlorid solution, 
50 per cent, glycerin solution, and many others. Frequently a strong 30 
per cent, potassium hydrate solution will enable the worker to obtain a very 
good idea of the presence or absence of red cells. Marx's fluid (made as 
follows, hydrochlorate of quinin 10 c.c. of a 1 to 1,000 solution added to 10 c.c. 
of 33 per cent, potassium hydrate solution, and tinted with eosin which will 
stain the erythrocytes a characteristic reddish hue) is a very excellent examining 
fluid. 

iSee Rous and Turner (Jour. Am. Med. Assn., 1915, LXIV, 1980), for an alternative 
method of testing the blood in such cases. 



THE BLOOD. 655 

As the presence of the red cells is so hard to demonstrate, search for them 
is frequently omitted and reliance placed more upon the demonstration of 
the hemoglobin in such stains. If the stain is upon a hard surface it may 
be scraped off with a clean piece of glass. If it be upon cloth, a portion of 
the stained part and also of the unstained part is removed and cut into small 
pieces with scissors which are absolutely clean. Such small pieces not broader 
than 1 mm. are very easily handled and can be readily teased out if desired. 

(2). Guaiac Test. 

This test originally devised by van Deen is one of our oldest for the detection 
of blood coloring matter, but seems to be much more reliable in its negative 
phase than when positive. The principle of the test is as follows: To a 
watery solution of the suspected stain is added an equal portion of fresh tincture 
of guaiac. This tincture is best made by dissolving the guaiac resin in alcohol 
when needed. The addition of the guaiac tincture to the suspected solution 
causes a milky turbidity. On now adding ozonized oil of turpentine, peroxid 
of hydrogen, or oil of eucalyptus, so that these latter substances float upon the 
guaiac and blood solution, a distinct blue color will be manifested at the point 
of contact, and, on shaking the tube, the coloration will spread throughout 
the mixture. This test seems to be very delicate, demonstrating the presence 
of blood in a dilution of several thousand. The blue coloration observed in 
this test is due to the oxidation of the guaiac to guaiaconic acid, which is then 
further oxidized into guaiac blue by the catalytic action of the oil of turpentine. 
It has been shown by Taylor that this reaction is also given by many substances 
among which we find manganate and permanganate of potassium, peroxid 
of manganese, peroxid of lead, chlorin, bromin, iodin, nitric and chromic 
acids, ferric chlorid, salts of copper, ferro- and ferricyanid of potassium. 
gum acacia, gluten, unboiled milk, raw potato pulp, pus, and any living cell 
or its intracellular enzymes. Various enzymes such as the oxidases will give 
this reaction. Buckmaster advises the use of the pure guaiaconic acid along 
with peroxid of hydrogen in the place of the guaiac resin and believes that the 
test increases in delicacy and accuracy under these conditions. He states that 
fluids containing hemoglobin or most of its derivatives give this test when it is 
impossible to detect pigments by any other methods and rightly adds that 
boiling the fluid suspected of containing blood does not interfere with the reac- 
tion but, on the contrary, throws out of consideration the action of milk, pus, 
fibrin, or of any enzyme. For the proper performance of this test the fluid to be 
tested, should not be alkaline and only very slightly, preferably not at all, acid. 

(3). Schaer's Test. 

This test is similar in many ways to the van Deen test, but employs, instead 
of guaiac, a 1 to 4 per cent, solution of aloin in alcohol. On adding this tincture 
to the suspected solution a red color soon becoming distinctly cherry-red appears 
upon the addition of ozonized oil of turpentine. Many other substances 
give a pink color, but only after the lapse of one or two hours, while the color 
with blood appears in a very short time. The tincture of aloin should always 
be freshly prepared, as of itself it undergoes this color change after standing. 



656 DIAGNOSTIC METHODS. 

(4) . Phenolphthalin Test. 

This test, 1 originally employed by Meyer 2 and, later, by Utz, 3 has been so 
improved by Kastle and Amoss 4 and Delearde and Benoit 5 that it is, probably, 
our most delicate one for the detection of blood, especially in stains, its limit of 
delicacy being about 1 part of blood in 8 million of water. As in the previous 
tests, the hemoglobin acts as an oxygen carrier, the active oxidizing agents being 
ozonized turpentine or hydrogen peroxid. The oxidases, peroxidases and 
catalases play small roles in this process in the case of blood-stains, but, in the 
various secretions and excretions of the body, their influence is very great. 

Phenolphthalin is a product of the reduction of phenolphthalein by zinc in 
alkaline solution. When oxidized in alkaline solution, it is converted into 
phenolphthalein with production of an intense red color. The reagent may be 
obtained in the market or, preferably, prepared as follows. Phenolphthalein 
is dissolved in a considerable excess of 30 per cent, sodium hydrate solution and 
boiled with an excess of zinc dust until a few drops of the strongly alkaline liquid 
no longer give a red color after neutralization with HC1 and sufficient alkali to 
alkalinize the solution. The solution is then decanted from the excess of zinc 
dust and the phenolphthalin is precipitated by acidifying with HC1. Collect 
, the precipitate on a filter and purify by repeated crystallization from water and 
alcohol. This purification is continued until a white crystalline compound is 
obtained free from every trace of phenolphthalein (as shown by absence of red 
coloration on addition of alkali). Dry at room temperature or in the oven at 
50 to 8o° C, care being taken to avoid contact with metallic surfaces. This 
compound should be kept in tightly stoppered bottles in a dark place, as oxida- 
tion gradually occurs. Rarely do we find that the coloration is sufficient to 
interfere with the tests, but in the delicate forensic tests it may be necessary to 
repurify a product which has stood for some time. 6 

The solution, as used in the test, is as follows : Mix a slight excess of phenol- 
phthalin, prepared as above, with 1 c.c. of N/10 sodium hydrate solution and 
a few c.c. of redistilled (from glass) water, shake thoroughly and filter. To 
the filtrate add 20 c.c. of N/10 NaOH solution, 0.1 c.c. of 3 per cent, hydrogen 
peroxid solution and make the mixture up to 100 c.c. This solution shows no 
trace of pink coloration when fresh, but gradually acquires a color, which may 
become so intense that the reagent can not be employed. In forensic work, use 
only freshly prepared solutions. 

To 1 part of the aqueous solution of the stain or of the secretion or excretion 
to be tested add 2 parts of the reagent and allow to stand for a few minutes. 
In the presence of blood a pink to red color appears, the intensity depending 
on the amount of blood present. This reaction is retarded by the extracts of 

1 See Kastle, Bull. 51, Hyg. Lab. U. S. P. H. & M. H. S., 1909. 

2 Munch, med. Wchnschr., 1903, L, 1489. 

3 Chem. Ztg., 1903, XXVII, 115 1. 

4 Bull. 31, Hyg. Lab. U. S. P. H. & M. H. S., 1906. 

5 Comp. Rend. Soc. de biol., 1908, LXW, 990. 

6 For routine laboratory work this reagent may be prepared as follows; Two grams of 
phenolphthalein, 20 grams of sodium hydrate, 10 grams of zinc dust and 100 c.c. of distilled 
water are mixed and heated until the solution becomes colorless. Filter while hot and preserve 
in an amber bottle. This solution is prone to show a slight reddish tinge from time to time 
owing to reoxidation, so that it may be necessary to heat it with zinc dust again and refilter. 



THE BLOOD. 657 

various animal tissues or various secretions of the body. For this reason we 
are never able to detect as small amounts of blood in the secretions as in watery 
solutions of pure blood, the limit of delicacy of all reactions being far less in the 
former than in the latter case. Boiling the solutions before applying the test 
removes most of the interfering factors. If the secretion be treated with a 
thick cream of aluminium hydrate suspension, the precipitate will carry down 
the blood pigment and thus concentrate it. A small amount of this precipitate, 
whether derived from saliva, urine, feces suspension, milk, or exudates, will 
show a decided red color when added to 2 c.c. of the reagent. Of course, in 
applying the test to the aqueous solution of the blood stain, no such treatment 
need be employed. 



: -^* iitf 3**n 

Fig. 158. — Hasmin Crystals from Human Blood. {Hawk.) 

Reproduced from a micro-photograph furnished by Prof. E. T. Reichert, of the 
University of Pennsylvania. 

This test is especially recommended for the detection of blood in all secre- 
tions and excretions (boiling them before applying the test) and, particularly, in 
the examination of suspected stains for forensic purposes. All possible fallacies 
must be guarded against and confirmatory tests used if necessary. 1 

(5). Teichmann's Test. 

This is one of the most important tests for the presence of blood and 
when positive is conclusive proof of the kind of stain with which one is working. 
A drop of blood or a portion of the suspected stain is spread upon a glass slide 
and covered with a drop of a very dilute solution (o.oi per cent.) of common 

1 Vas (Deutsch. med. Wchnschr., 1912, XXXVIII, 1412) calls attention to the possibility 
of the presence of phenolphthalein as a purgative (if feces be tested) . The addition of alkali 
alone will give a red color under this condition. See Cade and Mulsaut, Lyon Med., 1912, 
CXIX, 885; also de Jager, Berl. klin. Wchnschr., 1914, LI, 795; Schirokauer, Deutsch. med. 
Wchnschr., 1914, XL, 1472 and 1617; Schneider and von Teubern, Ibid., 1673; Boas, Ibid., 
1915, XLI, 549. 
42 



658 DIAGNOSTIC METHODS. 

salt. 1 The salt solution is then evaporated at a low temperature. A few drops 
of glacial acetic acid are then placed upon the salted stain and the preparation 
is covered with a cover-glass. The acid is now slowly evaporated by holding 
the slide over a flame in such a way that the fluid steams but does not boil. 
As the acid evaporates, more is allowed to run under the cover-glass, this addi- 
tion and evaporation being done thrice. The specimen is cooled and mounted 
in glycerin or distilled water, after which it may be examined with the dry 
lens. A successful specimen, which is not always obtained, will show the 
presence of numerous brownish rhomboid crystals, which are separate or grouped 
in sheafs or rosettes. These are the hemin crystals or hydrochlorate of hematin, 
called, after their discoverer, Teichmanri's crystals. 

It very frequently happens that the specimens are not successful, so 
that several slides should be made before a negative report is given. Reasons 
for failure may be found in the excessive heat applied to evaporate the acid, 
in the fact that the salt solution may have been too concentrated, or that the 
stain may have been very old. The addition of salt seems to be unnecessary 
except in the case of old blood stains or when the blood is poor in salts. It 
should even then be added in only very small amounts. As pointed out by 
Rose, when blood is mixed with iron rust the hemin test is usually negative. 2 

(6). Spectroscopic Examination of Blood. 

Certain characteristic appearances of the blood are noted on spectroscopic 
examination. The spectrum of the various blood pigments has been given in 
previous sections so that only a few additional remarks are necessary in this 
place. In spectroscopic examinations, the use of the ordinary hand spectro- 
scope is all that is necessary, the more complicated ones adding little to the 
differentiation of the various spectra. If the blood be fresh and the hemoglobin 
unaltered the spectrum is, of course, that of oxyhemoglobin; but as the usual 
stains submitted for examination are frequently very old ones, such a spectrum 
is practically never seen. The end-product of the alteration of hemoglobin as 
found in old blood stains is hematoporphyrin, which is iron-free and can be 
identified easiest by the spectroscope. The suspected stain is dissolved in 
concentrated sulphuric acid yielding a reddish-violet fluid, which is then examined 
spectroscopically. The spectrum of hematoporphyrin has been given previously. 
Many blood stains can be recognized only by this test, so that it is always 
advisable to make a spectroscopic examination, especially if other tests for 
hemoglobin have failed. 3 

(7). Precipitin Test. 

The precipitin test has been discussed in great detail previously (page 614). 
This is the absolute identification test for a stain, providing the stain has been 
shown to be a blood stain by the tests given above. The precipitin test is 

1 Nippe (Deutsch. med. Wchnschr., 1912, XXXVIII, 2222) advises one to mix the specimen 
with a few drops of a solution of 0.1 gram each of potassium bromid, iodid and chlorid in 100 
grams of glacial acetic acid. This modification shows up the crystals even when blood is 
mixed with rust See, also, Symons, Biochem. Jour., 1913, VII, 596. 

2 See Symons, Biochem. Jour., 1913, VII, 596; Beam and Freak, Ibid., 1915, IX, 161. 

3 See Sutherland and Mitra, Biochem. Jour., 1914, VIII, 128. 



THE BLOOD. 659 

specific, within very narrow limits, and is recognized in law as reliable in 
differentiating human blood from that of the domestic animals. Its one fallacy 
is that it reacts with the blood of anthropoid apes just as it does with that of 
man, but in most cases this element may be easily excluded. 

X. Value and Limitations of Blood Examinations. 

It has been truly said that the value of a blood examination -"is measured 
by the practical use which may be made of it, and not by any interesting yet 
useless information it may throw on the case." In clinical work we are prone 
to follow well-established lines of procedure and to forget, when engaged in 
counting the cells in an obscure case, that "the meaning and aim of the 
clinical study of the blood covers a much wider field than is embraced in 
the mere investigation of the histological elements." Yet our knowledge 
regarding the variations in the plasma is so meager that the busy practitioner 
may be pardoned for such neglect. 

There can be little question that the so-called "blood diseases" have 
the most characteristic variation from the normal, yet even here it is not so 
easy, as one might assume, to make an indisputable diagnosis in all cases. Thus, 
aside from the pure parasitic diseases, leukemia and advanced pernicious 
anemia seem to be the only ones capable of a certain diagnosis from the examina- 
tion of the blood. True the various anemias may be detected, but the etiology 
is not always clear nor the differentiation of a primary from a secondary form 
invariably possible. 

It is, of course, evident that bacteriologic examination of the blood and 
the application of the agglutination reaction may clear up many an obscure 
case, but such work requires much more time and much more attention to 
detail than is at the disposal of the busy oractitioner. It is, however, in just 
this class of infectious fevers that much benefit, in a confirmatory way, is forth- 
coming from a careful study of the blood. Thus, a leucocytosis or a leucopenia 
in any suspected case might lead to a differentiation of central pneumonia 
from typhoid fever or a scarlet fever from a case of measles. Moreover, a 
leucocytosis following a leucopenia in typhoid would strongly indicate perfora- 
tion and consequent surgical interference. Suppurative processes anywhere 
in the system are usually associated, unless very limited in extent, with a 
polynuclear neutrophile leucocytosis; so that the leucocyte count may be 
of great importance in judging of the extension of a pus infection. Immediate 
operation is occasionally decided upon after a leucocyte count, but this should not 
be made the only basis for such intervention unless frequent counts have given 
the surgeon the definite knowledge that a leucocytosis has suddenly occurred. 

It should be stated as an axiom that blood findings may never be interpreted 
except in the light of the clinical findings. To the clinician it should be said, 
never trust the laboratory report implicitly unless it agrees with the clinical mani- 
festations; to the laboratory worker ,we would say, never report a blood finding 
as diagnostic without knowing something of the clinical history of the case. 

A single blood examination will rarely be of any greater value than will 



66o DIAGNOSTIC METHODS. 

a single temperature determination. It is the series that enables one to decide 
either as to diagnosis or as to operative or therapeutic procedures. What 
we need most is not so much blood work, but much better work when done. 
Too much reliance is often placed on blood reports, so that a word of caution 
does not seem ill advised. Never rely solely on the blood findings, but use 
them merely as one of many links in your chain of clinical evidence. In this 
way mistaken diagnoses will occur less frequently. 

BIBLIOGRAPHY. 

i. Arneth. Die neutrophilen weissen Blutkorperchen. Jena, 1904. Diagnose 
und Therapie der Anamien. Wiirzburg, 1907. 

2. Besancon et Labbe. Traite* d'hematologie. Paris, 1904. 

3. Bramwell. Anemia and Diseases of the Ductless Glands. Edinburgh, 

1899. 

4. Buckmaster. The Morphology of Normal and Pathological Blood. Phila- 

delphia, 1906. 

5. Cabot. Clinical Examination of the Blood. New York, 1904. 

6. Canon. Bakteriologie des Blutes. .Jena, 1905. 

7. Celli. Malaria According to the New Researches. London, 1900. 

8. Champy. Le sang et les maladies du sang. Paris, 1913. 

9. Charles. Fonction des leucocytes. Paris, 1904. 

10. Coles. The Blood: How to Examine It. London, 1898. 

n. Craig. Estivo-autumnal Malarial Fevers. New York, 1901. The Malarial 

Fevers. New York, 1909. 
12 Da Costa. Clinical Hematology. Philadelphia, 1905. 

13. Decastello and Krjukoff. Untersuchungen iiber die Struktur der Blut- 

zellen. Wien, 191 1. 

14. Ehrlich, Lazarus, Pinkus, und von Noorden. Blut-Krankheiten. 

Wien, 1 913. Translation, Philadelphia, 1905. 

15. Ehrlich und Lazarus. Die Anamie. 3te Auf. Wien, 19 13. 

16. Ewing. Clinical Pathology of the Blood. Philadelphia, 1903. 

17. Gilbert et Weinberg. Traite du sang. Paris, 19 13. 

18. Grassi. Die Malaria. Jena, 1901. 

19. Grawitz. Klinische Pathologie des Blutes. Leipzig, 19 11. 

20. Hayem. Du sang. Paris, 1889. 

21. Helly. Die hasmopoetischen Organe. Wien, 1906. 

22. Jane way. The Clinical Study of Blood-pressure. New York, 1904. 

23. Kahane. Die Chlorose. Berlin, 1901. 

24. Kaplan. Serology of Nervous and Mental Diseases. Philadelphia, 1914- 

25. Kolmer. Serology. Philadelphia, 1914. 

26. Laveran. Du paludisme et de son hematozoaire. Paris, 1891. Traite* 

du Paludisme. Paris, 1907. 

27. Laveran et Mesnil. Trypanosomes and Trypanosomiases. Chicago, 

1907. (Translation by Nabarro.) 

28. Lenhardt. L'Anemie a type chlorotique. Paris, 1906. 

29. von Limbeck. Klinische Pathologie des Blutes. Jena, 1896. 

30. Mannaberg. Die Malariakrankheit. Wien, 1899. 

31. Naegeli. Blutkrankheiten und Blutdiagnostik. Leipzig, 19 n. 

32. Oliver. Studies in Blood-pressure. London, 1908. 

33. Pappenheim. Atlas der menschlichen Blutzellen. Jena, 1905 and 1912. 

Clinical Examination of the Blood and Its Technique. New York, 
1914. 



THE BLOOD. 66l 

34. Patella. I leucociti non granulosi del sangue. Torino, 1906. La genesi 

endoteliale dei leucociti mononucleali del sangue. Siena, 1907. 

35. Potain. La Pression arterielle de 1'homme. Paris, 1902. 

36. Prowazek. Handbuch der pathogenen Protozoen. 2te Aufl. Leipzig, 

1914-15- 

37. Reinert. Die Zahlung der Blutkorperchen. Leipzig, 1891. 

38. Ruge. Malaria Krankheit. Jena, 1906. 

39. Schleip. Atlas der Blutkrankheiten. Berlin, 1908. Translation, New 

York, 1908. 

40. Schoenborn. Gefrierpunkts- und Leitfahigkeitsbestimmungen. Wies- 

baden, 190/1. 

41. Stephens and Christophers. The Study of Malaria. London, 1905. 

42. Sternberg. Primarerkrankungen. Wiesbaden, 1905. 

43. Strauss und Rohnstein. Anamien. Berlin, 1901. 

44. Sutherland. Blood Stains. New York, 1907. 

45. Tallqvist. Ueber experimentelle Blutgiftanamien. Helsingfors, 1899. 

46. Thayer. Lectures on the Malarial Fevers. New York, 1897. 

47. Thayer and He wetson. Malarial Fevers of Baltimore. Baltimore, 1895. 

48. Thomas and Ivy. Applied Immunology. Philadelphia, 191 5. 

49. Turk. Vorlesungen ueber klinische Haematologie. Wien, 1904 and. 19 12. 

50. Uhlenhuth. Das biologische Verfahren zur Erkennung und Unterscheid- 

ung von Menschen und Tierblut. Jena, 1905. 

51. Ward. Bedside Hematology. Philadelphia, 19 14. 

52. Watkins. Diagnosis by means of the Blood. New York, 1902. 

53. Weiss. Haematologische Untersuchungen. Wien, 1896. 

54. Wile. Blood Examinations in Surgical Diagnosis. New York, 1908. 

55. Wolff. Die Kernzahl der Neutrophilen. Heidelberg, 1906. 



CHAPTER IX. 
TRANSUDATES AND EXUDATES. 

I. General Considerations. 

The serous membranes are normally kept moistened by liquids whose 
quantity is only sufficient in a few instances, as in the pericardial cavity and 
the subarachnoidal space, for a complete chemical analysis to be made of them. 
Under pathological conditions an abundant transudation may take place from 
the blood into the serous cavities, into the subcutaneous tissues or under the 
epidermis. If such conditions be the result of circulatory disturbance, the 
kidneys are usually unable to eliminate the normal amount of fluid from the 
system and, as a result, the retained fluid collects both in the serous cavities and 
in the areolar tissue. Such accumulations of fluid, known as transudates, 
are similar to the lymph, being, as a rule, poor in cellular elements and yielding 
little or no fibrin. These transudates must be sharply differentiated from the 
accumulations of fluid, which are the result of direct inflammatory processes 
in the membranes lining the serous cavities and are known as exudates. These 
latter fluids are generally rich in cellular elements and yield relatively more 
fibrin. As a rule, the richer a transudation is in leucocytes, the closer it stands 
to pus, while the poorer it is in these elements the closer it resembles true 
lymph. 

The formation of true transudates is largely a question of filtration under 
the influence of the rate of blood flow, the blood pressure, the irritation of 
the capillary endothelium, and the variable permeability of the endothelial 
cells. We should expect, therefore, that the passage of dissolved substances 
from the blood would be regulated by the same laws that control the secretion 
of physiologic fluids, namely the laws of passage of fluids through semipermeable 
membranes. The crystalloids would be, therefore, in approximately the 
same concentration as in the blood plasma, while the colloids must be in far 
less concentration, the actual values being influenced, of course, by the special 
membrane through which the fluid passes. The condition of the blood would 
hence affect the chemical composition of such transudates, hydremic plasma 
'yielding a fluid poorer in solids, while anhydremic blood is associated with a 
transudate of higher specific gravity. 

From a clinical standpoint a differentiation between transudates and 
exudates is not infrequently impossible, so that it is advisable to resort to 
aspiration of the fluid and to chemical and microscopical examination of the 
material withdrawn. The chief phases of such examinations are: (i) the 
chemical and physical properties of the fluid; (2) the bacteriological aspect 
of the fluids, and (3) the morphological characteristics of the cellular elements. 

662 



TRANSUDATES AND EXUDATES. 663 

Obtaining the Specimen. 

Whenever fluid is to be withdrawn, either for diagnostic or therapeutic 
purposes, it is necessary to resort to puncture of the cavity containing the fluid. 
In all cases the site of puncture must be as carefully prepared as in any surgical 
procedure. Puncture may be performed with the ordinary trocar or, preferably, 
with a large needle which has a rather large lumen. The instruments must 
be carefully sterilized before use. If the skin is especially tough, it is advisable 
to make a small incision through the skin and insert the needle through the 
incision. Very little pain is felt by the patient during this procedure, but 
the writer is accustomed invariably to resort to the use of ethyl chlorid to 
anesthetize the part. 

In the withdrawal of pleuritic effusions the spot selected should be neither 
too high nor too low. It may be in the seventh intercostal space in the 
axillary line or in the eighth intercostal space at the outer angle of the scapula. 
The arm of the patient should be brought forward with the hand resting on the 
opposite shoulder, in order to widen the intercostal spaces. In inserting the 
needle it is wise to make the thrust close to the upper margin of the rib so as to . 
avoid the intercostal artery. In all cases the fluid should be withdrawn slowly 
and the excess above that required for examination allowed to drain until the 
desired amount is obtained. If the puncture is for diagnostic purposes, 10 to 20 
c.c. are sufficient, while the therapeutic withdrawal of fluid will vary with the 
amount present and with the clinical symptoms of the case. Should the patient 
show signs of shock or of depression during the operation, the procedure should 
be interrupted as quickly as possible. Aspiration is rarely necessary or advisable. 

In the withdrawal of fluids from the abdominal cavity, the needle or 
trocar is thrust through the lower abdominal wall and the fluid collected. In 
this procedure more or less danger of puncturing the bowel exists if the 
effusion be small, so that the needle should not be carelessly inserted lest this 
complication arise. Naturally, in well-marked ascites no such danger is present. 

II. Physical and Chemical Properties. 

Transudates are, as a rule, serous in character, usually transparent, colorless, 
or light yellow in color, but at times showing a milky, reddish, or a greenish 
tinge, the latter practically always being observed after the fluid has stood 
exposed to the air: Such solutions are, as a rule, dichroic, yellow by transmitted 
light and green by reflected light. They are alkaline in reaction and show a 
specific gravity, which varies, according to the origin of the fluid, from 1,006 to 
1,018, while serous exudates from the same cavities show a much higher specific 
gravity. 1 The variations in specific gravity depend largely upon the amount of 
albumin present in the transudate, this practically never being over 3 per cent, 
and usually much lower. The chief proteins present are albumin and globulin, 
these being related to one another in the transudates as one and one-half to one, 
while in the exudates the globulin is relatively much increased. 2 The determina- 

1 See Trevisan, Ztschr. f. exper. Path. u. Therap., 1912, X, 141. 

2 See Mosny, Javal and Dumont, Presse mid., 1914, XXII, 477; Ujihara, Biochem. 
Ztschr., 1914, LXI, 55; Berl. klin. Wchnschr., 1914, LI, 1112; Villaret, Jour, de Physiol, et de 
Path, gen., 1913, XV, 617, 652 and 875. 



664 DIAGNOSTIC METHODS. 

tion of the total protein may be made by methods of fractional precipitation as 
previously discussed. The transudates from the pleura contain the largest 
percentage of albumin, while edematous fluids rarely show over i per cent. 
Transudates do not coagulate spontaneously. Glucose is present both in 
transudates and exudates in amounts varying between 0.04 and 0.1 per cent. 
The mineral constituents of transudates are somewhat higher than in the 
exudates, the former averaging 0.96, the latter 0.89 per cent. Under patho- 
logical influences fat, blood, uric acid, and biliary pigment may find their way 
into both types of fluid. In diabetes an excess of sugar and the presence 
of acetone bodies may be detected. 

The exudates are usually straw- or lemon-yellow in color depending on 
the degree of inflammation, or they may assume colorations ranging from a 
deep red (hemorrhagic) to a milky (purulent) shade. Biliary pigments may 
cause a bright green, while various medicaments, such as methylene blue, may 
produce a greenish-blue coloration. The specific gravity is almost invariably 
above 1,018, the reaction is alkaline, the albumin content is usually above 
3 percent., reaching as high as 7 per cent., while the globulin is relatively 
much increased in comparison with the albumin. This globulin increase is 
largely due to paraeuglobulin. Nucleoprotein is especially abundant in 
purulent exudates in which the autolytic processes are more or less marked. 
The total nitrogen of the various fluids varies from 0.22 to 1.38 per cent. The 
nitrogen partition may be seen from the following table of Gerhartz. 1 Exudates 
coagulate spontaneously on standing. 2 



Figures in terms | Total 
of % (grams per 

100 c.c.) N 



Precipi- Protein Ammonia Purin 

table 

N N N N 



Urea Amino-acid 
N N 



Transudate 1 0.22— 0.58 0.18— 0.53 0.17—0.52 0.007-0.01 0.002—0.007 0.01-0.05 0.002-0.005 

Serous exudate. ... jo. 43— 1 .09 0.39— 1 .07 0.37— 1 .03 o. 01 —0.03 0.01 0.01—0.06 0.007 

Purulent exudate. . 1 .11-1 .33 0.94— 1 .22 1.14 0.01 0.007 0.02-0.1 0.004—0.28 

The exudates, which, accurately speaking, are always of inflammatory 
origin, may be serous, serofibrinous, seropurulent, , hemorrhagic, purulent, 
putrid, chylous, and chyloid. 
Serous Exudates. 

These are clear, of a light straw color, and show a specific gravity above 
1,018. There is a large amount of fibrin, as shown by the dense network 
microscopically, containing a few red cells which may be derived from the 
bleeding at the point of puncture, a few leucocytes which may vary in type 
according to the kind of bacteria causing the infection, and large endothelial 

1 Chemie der Transudate and Exsudate, Jena, 1908. See, also, Wiener (Biochem. Ztschr., 
1912, XLI, 149) regarding the presence of proteolytic ferments and amino-acids in exudates; 
and Lenk and Pollak, Deutsch. Arch. f. klin. Med., 1913, CIX, 351. See Hegler and 
Schumm, Med. Klin., 1913, IX, 1810; Dorner, Deutsch. Arch. f. klin. Med., 1914, CXIII, 
342. 

2 Rivalta advocates the following test to differentiate an exudate from a transudate: 
In a conical glass place about 200 c.c. of water and add 2 drops of glacial acetic acid. Mix 
thoroughly and allow 1 or 2 drops of the puncture-fluid to fall into this weak acid solu- 
tion. If the fluid be an exudate a distinct cloud will be observed in the wake of the falling 
drop, while if it be a transudate little or no turbidity will be noticed. This reaction is 
due to the large globulin content of the exudate. 



TRANSUDATES AND EXUDATES. 665 

cells from the serous membrane lining the cavity. If the blood-cells be present 
in sufficient numbers to give a distinct red color to the fluid it is termed a hemor- 
rhagic exudate, while if a few pus-cells are found it may be called a seropurulent 
type. The gradations between the true serous and serofibrinous types of 
exudate are exceedingly varied, so that it is difficult to tell which type is really 
present. Even from a purely serous exudate a certain amount of coagulation, 
with formation of a distinct fibrin network, may be obtained, so that the only 
criterion would be one of degree. 1 

The type of leucocyte present is usually of the polymorphonuclear variety, 
although other forms may be present. It is, therefore, an important part 
of the examination of an exudate to determine the percentage relations of these 
various cellular elements. This will be discussed in the section on Cytology. 

Chylous Exudates. 

On account of the close relationship between the abdominal, thoracic, 
and pericardial cavities, on the one hand, and the large lymphatic trunks, on the 
other, it is possible for lymph to pass directly into these cavities in case rupture 
of these vessels occurs. Such exudates show all the properties of chyle. The 
fluid is white and milky, contains between 1.5 and 2.5 per cent, of protein, and 
a considerable amount of fat, which may be demonstrated by staining with 
osmic acid or Sudan-Ill or by alkalinizing with sodium hydrate and shaking 
out with ether. 

Chyloid Exudates. 

It has been found that carcinoma, tuberculosis, extreme cardiovascular 
changes, hepatic disturbances, puerperal sepsis, and infection with the filaria 
may give rise to a chyloid type of ascites. The fluid in these cases is less milky 
than that of the true chylous type, contains less fat, and is not so completely 
cleared by shaking with ether. It contains serum-albumin, mucin and, es- 
pecially, rather large amounts of a complex of pseudoglobulin and lecithin, to 
which the opacity is due. 2 Traces of sugar are found but not as large in 
amount as in the true chylous types. These fluids resist putrefaction for long 
periods owing to their lecithin content. The accumulation of fluid after tapping 
is much more rapid in the chylous than in the pseudo-chylous types. 

Hemorrhagic Exudates. 

This type is, in reality, a serofibrinous form containing large numbers 
of blood-cells. It is observed in patients with hemorrhagic diathesis, in 
connection with active tuberculosis, with neoplasms of the serous cavities, 
and following injuries to the chest or abdomen. In this form of exudate, the 
exciting bacterial agent, usually the tubercle bacillus, may occasionally be 
found, but not always. The type of leucocyte (mononuclear) would be very 
strong presumptive evidence in favor of tuberculosis, even though no bacilli 
were found. If the exudate be due to a malignant growth, it is sometimes 
possible to obtain shreds of the tumor tissue and thus make a probable diagnosis. 

1 See Epstein, Jour. Exper. Med., 1914, XX, 334. 

2 See Waliis and Scholberg, Quart. Jour. Med., 19 10, III, 301; Ibid., 19 11, IV, 153; also 
Henry, New York Med. Jour., 1911, XCTV, 1; and Lecliard, Brit- Jour. Child. Dis., 1913, X, 
433; Fubini, Gazz. d. osp., 191 5, XXXVI, 145. 



666 DIAGNOSTIC METHODS. 

In judging of the malignancy of the cells, it is sometimes difficult to differentiate 
the abnormal from the normal, especially in the affections of the pleura. These 
malignant cells are usually extremely large and are characterized by their 
vacuolation and fatty degeneration (see cut). It is not infrequent to find 
in hemorrhagic exudates, which have remained in the body cavity for some time, 
a large number of cholesterin crystals and occasionally small masses of 
hemosiderin. 

Purulent Exudates. 

These are composed either of true pus or of seropus. They are more or 
less yellow in color, thick and occasionally tenacious, and separate on standing 
or centrifuging into a cellular deposit and a pus serum. The cells forming 
the pus are not infrequently in a condition of advanced fatty degeneration 
and may contain numerous bacteria. The addition of acetic acid will usually 
clear up the cells so that the nuclei become recognizable. Fat is present in 
amounts as high as 7 per cent., while a relatively large amount of various pro- 
teins and extractives is shown on chemical examination. Fatty acid crystals 
and cholesterin may be abundant in the sediment, especially in old abscesses. 
Fresh pus is usually alkaline, but may become distinctly acid, owing to the 
development of lactic acid in the process of autolysis. 

Purulent exudates are investigated with special regard to the type of cell 
present and to the organism associated with the pus formation. In ordinary 
pus, the cell is of the polymorphonuclear type, although occasionally the 
mononuclear form may be predominant. The bacteria are very numerous 
and include many of the most important types. While the pyogenic bacteria 
are more frequently the various types of staphylococci and streptococci, it is 
to be remembered that other organisms may produce pus under certain condi- 
tions. Thus the typhoid bacilli, colon bacilli, pneumococci, Friedlander's 
bacilli, gonococci, diphtheria bacilli, Morax-Axenfeld and Koch-Weeks bacilli, 
and influenza bacilli, among others, may be the causative factor in the formation 
of a purulent exudate. It will be seen, therefore, that the bacterial examination 
of a purulent exudate may require a very extensive research. As it is frequently 
impossible to decide, from a stained specimen, as to the special organism 
causing the infection, cultures should be made in every doubtful case. The 
peculiarities of such cultures may be found in any work on bacteriology. 

Putrid Exudates. 

These may be observed in various cavities of the body or in the substance 
of various organs, especially the liver and lungs. They arise from the entrance 
of pus into these cavities from perforation of a gangrenous area, gastric or 
intestinal ulcer, malignant growths, etc. The material obtained by puncture 
is usually brownish or greenish in color, has a very offensive odor, and is usually 
alkaline, but may be acid. Microscopically degenerated cells, numerous 
bacteria, cholesterin, fatty acid, and hematoidin crystals are observed. In 
some cases various portions of an echinococcus cyst may be found in the exu- 
dates. Bilirubin crystals and various ammo-acids may be found in rupture 
of an hepatic abscess. 



TRANSUDATES AND EXUDATES 667 

III. Bacteriology. 

It is always advisable to make cultures of the material obtained by puncture 
in order to discover the organism which is acting as the exciting cause of the 
condition under investigation. Usually a few c.c. of the fluid are allowed 
to drop into a flask containing 50 c.c. of sterile nutrient bouillon and the mixture 
incubated for 24 to 48 hours. From the growth, obtained in this preliminary 
work, subcultures are made on various media and microscopical examination 
employed to identify the organism. It is always advisable to make at least 
two microscopical specimens, staining one with the ordinary methylene blue 
stain and the second with Gram's stain. 

Tubercle Bacilli. 

If these organisms are suspected, the technic of staining is the same 
as outlined under Sputum. It frequently happens, in the examination of 
suspected tubercular exudates, that the presence of a large amount of fibrin, 
either with or without spontaneous coagulation of the specimen, makes it 
exceedingly difficult to find the organism, even though it be present. In such 
cases, advantage is taken of a procedure, recommended by Jousset, 1 known 
as inoscopy. If the fluid has not coagulated, it is allowed to do so in order that 
the coagulation may enclose the bacilli within the fibrinous network. The 
coagulum is separated as completely as possible from the supernatant fluid, 
is washed with distilled water, and is treated with 30 to 40 c.c. of the following 
mixture, which will digest the fibrin. 

Pepsin, 2 grams. 

Sodium fluorid, 3 grams. 

Glycerin, 10 c.c. 

Hydrochloric acid (cone), 10 c.c. 
Distilled water, q.s., ad., 1000 c.c. 

This mixture is placed in the incubator for 24 hours, when the fluid becomes 
homogeneous. The digested fluid is centrifuged and smears are made from 
the sediment as previously described. It is always advisable to treat such 
smears with a small quantity of albumin fixative, so that the organisms may 
not be washed from the slide. The smear is fixed in the flame and stained in 
the usual way with carbol fuchsin. The tubercle bacilli may not appear as 
deeply stained after such treatment. This method has been replaced, to a 
large extent, by the Antiformin method (see p. 18). 

Gonococcus. 

The gonococcus appears in stained specimens as small biscuit-shaped or 
coffee-berry-shaped cocci, which are arranged in pairs separated by a narrow 
unstained portion. Occasionally two of these pairs of hemispheres are joined 
together, forming tetrads. They may be stained by any of the anilin dyes, but 
are recognized especially by their reaction toward Gram's method. In this con- 
nection it must be said that other organisms, among which we find the diplococcus 

1 Semaine Med., 1903, XXIII, 22. 



668 DIAGNOSTIC METHODS. 

intracellularis meningitidis of Weichselbaum and the micrococcus catarrhalis, 
resemble the gonococcus both in morphological and staining characteristics, so 
that a differentiation by this method is not always possible. Fortunately, how- 
ever, the cultural peculiarities of these organisms absolutely differentiate them. 1 

Gram's Stain. 

The principle of the staining of various organisms by Gram's method 
is that certain organisms retain the primary color after treatment with decolor- 
izing agents, while others lose this primary stain and must be treated with a 
contrast stain for their recognition. If the organism retains the primary blue 
color it is called Gram-positive, while if it lose the primary color and take on 
the contrast stain it is called Gram-negative. Among the organisms which are 
Gram-negative we find the gonococcus, meningococcus, micrococcus catarrhalis, 
influenza bacillus, typhoid bacillus, colon bacillus, Koch- Weeks bacillus, and the 
Morax-Axenfeld bacillus; while the tubercle bacillus, smegma bacillus, diphtheria 
bacillus, pneumococcus, streptococcus, staphylococcus, and various saprophytic 
cocci found in the smears both of the male and female urethra are Gram-positive. 

The solutions required in Gram's method are: (i) an anilin oil — gentian 
violet mixture, consisting of 84 c.c. of anilin water (water saturated with anilin 
and filtered), and 16 c.c. of a saturated alcoholic solution of gentian violet; 
(2) a solution of iodin consisting of one gram of iodin, 2 grams of potassium 
iodid, dissolved in 300 c.c. of water; (3) a dilute solution of carbol fuchsin, 
a dilute aqueous solution of safranin, or a 1 per cent, aqueous solution of 
Bismarck brown as a contrast stain. 
Technic. 

Smears of the exudate are made, in the manner previously described 
for making blood smears, by receiving a drop of the purulent material upon 
one end of a glass slide and spreading it in a thin even layer by means of a 
second slide. These smears are then fixed by passing several times through 
the flame. When the slides have cooled, they are covered with the gentian- 
violet solution which is allowed to act for one to three minutes. The solution 
is then poured off and the excess removed by washing in water. Without 
drying, the iodin solution is placed on the slide and allowed to act for one-half 
to one minute. It is then washed in water and the preparation treated with 
95 per cent, alcohol until no more color is removed by it. The alcohol is 
then removed by washing with water and the smear is covered with one of the 
contrast stains above mentioned, in the writer's laboratory the safranin solu- 
tion, which is allowed to act for only a few seconds. The stain is then washed 
off with water and the slide dried between folds of filter-paper. 

The specimen under the microscope shows the Gram-positive organisms 
stained deep blue, while the Gram-negative bacteria and the bodies of the pus- 

1 See Warden (Jour. Infect. Dis., 1913, XII, 93), who emphasizes the importance of a 
slightly acid reaction in the media containing preferably human body fluids, such as ascitic or 
hydrocele fluid. Warden (Jour. Infect. Dis., 1913, XIII, 124) calls attention to the fact that, 
oftentimes, what appears to be the gonococcus in smear preparations is shown to be a staphy- 
lococcus on culture. This awaits confirmation. See, also, Whitney, Boston Med. and Surg. 
Jour., 1914, CLXX, 749; France, New York Med. Jour., 1914, C, 1255; Wolbarst, Ibid., 1915, 
CI, 146; Warden, Jour. Infect. Dis., 1915, XVI, 426; Pearce, Jour. Exper. Med., 1915, XXI, 
280. 



PLATE XXXIII. 



>L 







# 



%® 



% 

*' ^ 



XatV\ar.n« Hill. 



Gonococci in Urethral Discharge. (Gram's Stain.) 



TRANSUDATES AND EXUDATES. 669 

cells take the red safranin stain. In such specimens the gonococcus, if pres- 
ent, will be observed both intracellularly and extracellularly, the former being 
the more characteristic. The ordinary pus cocci may likewise be intracellular, 
but these are distinctly Gram-positive, while the gonococcus is Gram-negative. 
In the purulent exudate from the urethra, large masses of mucoid material 
may be present, which are known as gonorrheal threads. These may be found 
in the urinary sediment and are usually easily recognized. They contain 
masses of pus cells, within which may be found numerous gonococci. These 
shreds may persist for years in anyone with a history of a previous gonorrhea, 
but they may then contain no organisms. The cytology of gonorrheal pus 
presents nothing characteristic beyond the presence of numerous eosinophiles 
with large numbers of polynuclear neutrophiles and an occasional lymphocyte. 

Smegma Bacilli. 

In the exudate of the preputial follicles, known as smegma preputii, are 
found fat globules, ammonium soaps, cholesterin crystals, and a few epithelial 
cells. In this smegma are found many bacilli, smegma bacilli, which show 
the same morphological and practically the same staining characteristics 
as the tubercle bacillus. This is differentiated, as described under Sputum. 
If the urine is to be examined for tubercle bacilli, it is much better technic 
to use a catheterized specimen than it is to resort to methods of questionable 
differentiation. If this is done any acid and alcohol-fast bacilli may be regarded 
as the tubercle bacilli, but in all cases the smegma bacilli must be excluded. 1 

Ducreyi Bacillus. 

This bacillus is the organism causing soft chancre and is known as the 
bacillus ulceris cancrosi. It is found in the purulent discharge from the 
chancre, but rarely, if ever, in pure culture. The sections of tissue may, how- 
ever, show none but this organism. 

In making preparations for study of this organism, the ulcerated surface 
is scraped with a platinum loop, the pus spread upon a slide, dried in the 
air, and fixed with alcohol-ether or over the flame. It stains readily with 
all bacterial stains, but decolorizes with Gram's stain. 

In stained specimens the organism appears as a short, thick, oval bacillus 
with rounded ends and two lateral indentations, which occasionally give it the 
appearance of a figure 8. Ordinarily the extremities are more deeply stained 
than the central portion, this appearing almost clear. The organism has 
a tendency to form chains or groups, which are rarely found in the pus-cells 
but are frequently seen within the epithelial cells. 

Spirochaeta Pallida. 

The Treponema Pallidum, discovered by Schaudinn and Hoffmann, 2 is 
now definitely established as the causative factor of syphilis. It may be 
obtained from the primary chancre, the incised papules, condylomata, mucous 
patches, inguinal glands, as well as from the internal organs in tertiary and 

1 See Brereton and Smith, Am. Jour. Med. Sc, 1914, CXLVTII, 267. 

2 Arbeiten aus der kais. Gesundheitsamte, 1905, XXII, 527. 



670 DIAGNOSTIC METHODS. 

congenital syphilis. Its size, form, type of motility and method of cultivation 
have been discussed under blood. 

To obtain this organism from the primary lesions, the chancre is thoroughly 
cleansed either with normal salt solution or with soap and water, rinsed with salt 
solution and dried. If the sore is healed, the epithelial covering is removed. 
The chancre is lightly curetted or the edges scraped, the blood, which should 
be avoided, is wiped away and the serum which exudes used for the later work. 
A drop of this serum is placed upon a slide, covered with a cover-glass and ex- 
amined at once under the dark-field illuminator, the spirochete appearing as 
colorless, glistening, rapidly motile spirals. ' If this method, which is to be prefer- 
red to any staining method, is not possible, a thin smear is made from the serum, 
transferring a drop by means of a platinum loop to a slide and using a rapid circu- 
lar motion in spreading. The specimen is dried in the air and stained as follows. 1 

Giemsa's Stain. 

The air-dried film is fixed with chemically pure methyl alcohol and stained 
with Giemsa's stain (p. 483) for 20 hours, using 1 drop of the stain to 1 c.c. of 
water. The spirochete are stained a violet-red color, the refringens being blue. 

Ghoreyeb's Method. 2 

The smears are made as above and air-dried. Cover the smear with 
1 per cent, aqueous solution of osmic acid for 30 seconds. Wash thoroughly in 
running water. Cover with solution of lead subacetate (liquor plumbi subace- 
tatis diluted 100 times) for 10 seconds. Wash in water. Cover with 10 per 
■ cent, aqueous solution of sodium sulphid for 10 seconds. Wash in water. The 
above process is repeated three times. Following this the osmic acid solution 
is applied for 30 seconds, the specimen is washed in water, dried and mounted 
in balsam. The spirochete, bacteria and cellular detritus are stained black. 
This method is quick, reliable, and is to be recommended. 

India Ink Method. 

Burri 3 has advanced this method, which is simple and reliable, although 
some specimens of ink may show confusing artefacts according to Barach, 4 
who used an inferior ink. A drop of the fresh serum from the lesion is placed 
at one end of a glass slide and immediately mixed with a small drop of Gunther- 
Wagner India ink (Chin-Chin liquid pearl ink). The mixture is then spread 
and allowed to dry. The specimen is studied with the immersion lens. The 
whole field is a homogeneous brown or black color, the treponema, blood cells, 
etc., appearing as colorless highly refractile bodies. The morphology, length 
and numbers of spirals should distinguish the pallida from the other types. 5 

1 Tunnicliff (Jour. Am. Med. Assn., 19 12, LVIII, 1682) calls attention to a very simple 
and reliable method formerly used by Oppenheimer and Sachs as well as by Ploeger. Make 
smears as usual, fix in the flame and cover for a few seconds with a 10 per cent, mixture of a 
saturated alcoholic gentian-violet solution in 5 per cent, phenol. Wash in water and dry. 

2 Jour. Am. Med. Assn., 19 10, LIV, 1498. 

3 Das Tuschverfahren, Jena, 1909. See, also, Walters, Jour. Am. Med. Assn., 1914, 
LXIII, 1666. .'■••... . 

4 Jour. Am. Med. Assn., 1910, LV, 1892. 

5 Goosmann (Jour. Cutan. Dis., 1911, XXIX, 628) has advised the use of nigrosin as a 
substitute for India Ink. 



PLATE XXXIV. 






Katharlhe HUY 



Spirochete Pallida in Tissue. (Levaditi's Stain.) 



TRANSUDATES AND EXUDATES. 67 1 

Levaditi and Manouelian's Method. 

This method, l also known as the pyridin method, is applied only to the de- 
tection of treponema in tissues. It is superior to the older method of Levaditi. 
Cut the tissue into pieces about 1 to 2 mm. square, fix in 10 per cent, formalin 
for 24 hours and harden in absolute alcohol for 24 hours. Wash in distilled 
water until the pieces fall to the bottom of the vessel. Place the tissue in a 
ground-glass stoppered bottle and add 50 c.c. of the following mixture. 

1 per cent, aqueous solution of silver nitrate, 90 c.c. 
Pyridin, 10 c.c. 

Leave them in the silver solution for two to three hours at room temperature 
and for three to five hours at 50 C. Wash in 10 per cent, pyridin solution. 
Immerse for three to four hours at room temperature in a freshly prepared 
solution with following formula: 

4 per cent, aqueous solution of pyrogallol, 90 c.c. 

Pure acetone, 10 c.c. 

Pyridin, 15 c.c. 

Remove from the reducing bath, dehydrate in absolute alcohol, clear in xylol 
and embed in paraffin. Cut sections and stain on the slide with a 2 per cent, 
aqueous solution of toluidine blue. Differentiate in absolute alcohol or Unna's 
ether-glycerin mixture, dehydrate in absolute alcohol, oil of bergamot and xylol. 
Mount in balsam and examine. The spirochaete are stained a beautiful black, 
while the ground-work of the tissue is blue. 

Noguchi's Method. 

This method 2 is used with beautiful results, especially with brain or cord 
tissue. Harden the tissue in 10 per cent, formalin for several days. The 
longer this is continued the better the results. Place the pieces (5 to 7 mm. 
thick) in the following mixture for five days at room temperature. 

Formalin, 10 c.c. 

Pyridin, 10 c.c. 

Acetone, 25 c.c. 

Alcohol (absolute), 25 c.c. 

Distilled water, 30 c.c. 

Wash thoroughly in distilled water for 24 hours. Transfer to 96 per cent, 
alcohol for three days and, then, wash in distilled water for 24 hours. Place 
the tissue in an amber bottle and treat with 1.5 per cent, aqueous solution of 
silver nitrate for three days at 37 C. or five days at room temperature. Wash 
in distilled water for several hours. Reduce in the following mixture for 24 
hours. 

4 per cent, aqueous solution of pyrogallol, 95 c.c. 

Formalin, 5 c.c. 

1 Compt. rend. Soc. de biol., 1906, LX, 134. See, also, Sobernheim, Kolle and Wassermann's 
Handbuch d. pathog. Mikroorg., 1913, VII, 770. 

2 Jour. Cutan. Dis., 19 13, XXXI, 547. 



672 DIAGNOSTIC METHODS. 

Wash in distilled water, transfer to 80 per cent, alcohol for 24 hours, then to 
95 per cent, alcohol for three days and absolute alcohol for two days. Clear 
in xylol, xylol-paraffin and embed in paraffin. Cut sections from various strata 
of the tissue. Sections 3 to 5 ^ give the best results. The treponemata are 
pure black in color, while the tissue varies from a pale yellow to a yellowish- 
brown. The neuroglia fibers sometimes stain distinctly, but are brownish, 
never black. 

IV. Cytology. 

The cytology 1 of transudates and exudates has reference to the study 
of the various types of cells found in such fluids. As a rule, such investigations, 
known as cytodiagnosi.s, are carried out more frequently on the nonpurulent 
types of fluid, as the examinations of the purulent fluids are more especially 
concerned with the bacteria present. 

Technic. 

The technic of obtaining the cellular components of the puncture fluid 
will vary according as the effusion does or does not contain fibrin. If this be 
present in fairly large amount, the fluid must first be defibrinated before the 
next steps are possible. Here, again, the procedure may be complicated by 
the presence or absence of coagula. If the fluid be not coagulated, it is placed 
in a large sterile flask which contains a few sterile glass beads, the mixture 
is actively shaken until a firm clot is obtained, in the meshes of which a few 
of the cells will necessarily be enclosed, but the majority will remain sus- 
pended in the liquid. The fluid is then separated from the clot and placed 
in centrifugal tubes which are drawn out to a rather fine point. If the fluid 
be coagulated when collected, it should be shaken with glass beads to break 
up the clot and liberate the cellular elements. The fluid is then separated 
and treated as follows. 

The remaining portion of the technic is the same for fluids which 
contain or do not contain fibrinous material. The centrifuge is rotated rather 
rapidly for about ten minutes in order to collect the cells as a sediment. In 
most of the exudates the number of these cells is very small so that a concen- 
tration is absolutely essential. As Widal and Ravaut have shown, the polynu- 
clear cells seem to be somewhat more affected by the defibrinization than the 
other types, so that these cells may show a relative diminution. After the 
cells have collected, the fluid is removed by rapidly inverting the tube in such 
a way that the sediment does not follow the liquid. Some workers advise 
the withdrawal of the sediment by a pipet with a long fine tip, but the writer 
has not found this method any more advantageous than the one spoken of 
above. The sediment in the tube is shaken so as to mix it thoroughly and a 
drop placed upon the glass slide and spread as described under Blood. Where 
very few cells are present it is wise to allow the drop to dry on the slide without 
spreading, in order to concentrate the cells in a small area. The specimen is 
then treated with Wright's stain and examined under the immersion lens. If 

x See Labbe, Cytodiagnostic, Paris, 1903; Ravaut, Cytodiagnostic, Paris, 1901; Brion, 
Centralbl. f. allg. Path., 1903, XIV, 609. 



TRANSUDATES AND EXUDATES. 673 

stains are to be used which contain no fixative, such as methyl alcohol, it is 
necessary that the specimen be fixed by methods previously discussed in the 
section on Blood. The stain to be used will depend largely upon the points 
to be studied, the eosin-hematoxylin stain being especially serviceable in 
differentiating the nuclear structures of the cell. As the cells in the various 
pathologic fluids frequently show more or less degeneration, the nuclear portion 
is more suitable for study than is the cell protoplasm. For this reason the 
writer prefers the use of the eosin-hematoxylin method along with the Wright 
stain or the triacid stain for the granules of the cells. 

These specimens may also be used for the study of the bacteria present. 
When this object is to be subserved, it is advisable to make two specimens, 
staining one with the ordinary methylene blue stain and a second by Gram's 
stain. If the material be very limited in amount, it is possible to combine the 
above staining methods by treating first with the eosin-hematoxylin method, 
washing in water and then following the ordinary procedure of the Gram 
method of staining. Such specimens are extremely panoptic and are especially 
to be recommended. 

Cytology of Normal Fluids. 

The number of cellular elements in fluids from the various serous cavities 
of the body may vary from a very few to a large number. The cells observed 
are the red and white corpuscles of the blood, the latter of which are usually 
relatively more numerous than in the circulating blood and are usually largely 
of the polynuclear type, although mononuclear forms are frequently present. 
Neutrophiles and eosinophiles are present under normal conditions, the latter 
being relatively more abundant than in the blood. If a large number of red 
cells are found, an injury of the small vessels during puncture usually explains 
their presence. Besides these types of cell, which are exactly similar to those 
of the blood, a few endothelial cells are practically always seen. These cells 
are observed of different shapes, may be single or grouped in sheets, and may 
be very much degenerated. They are larger than the other cellular elements, 
their contour is usually circular, but may be irregular; they are mononuclear, 
and frequently contain round vacuoles. 

In examining normal as well as pathologic fluids for their cellular content, 
100 cells should be counted if possible and the percentage of each type thus 
determined. This constitutes the cytologic formula of the exudate. 

Cytology of Pathologic Fluids. 

According to the theory of Metchnikoff, the presence of a bacterial in- 
fection is associated with attraction of the leucocytes to the infected area. 
These cells then enter into combat with the bacteria and either destroy the 
organism or are destroyed by them. As has been shown, certain organisms, 
especially the tubercle bacillus and probably the typhoid organism, attract 
the lymphocytes, while most of the other organisms attract the polymorphonu- 
clear neutrophiles. Theoretically, therefore, it should be possible to decide 
as to a tubercular or nontubercular condition by the presence or absence 
43 



674 DIAGNOSTIC METHODS. 

of an increased number of the mononuclear types of leucocytes. This is 
the basis of the attempt at differential diagnosis by means of cytodiagnosis. 
Not infrequently one finds in malignant conditions the so-called specific 
cells which are either sarcomatous or carcinomatous. These, although specific, 
are not easy absolutely to identify. These cells are very large, frequently 
showing fatty degeneration, extensively vacuolated, and showing a mitotic 
mulberry-like nucleus. Although differing from the endothelial cell, confusion 
is very apt to arise, so that it is difficult to make a diagnosis in all cases from 
the appearance of such cells. 

Pleural Exudates. 
Primary Tubercular Pleurisy. 

This is characterized especially by an increase, both relative and absolute, 
in the number of lymphocytes. A pleural lymphocytosis exists when there 
is an excess of mononuclear cells, with abundant protoplasm, a large nucleus, 
and smaller than the endothelial cells. In the very early days of the infection 
a neutrophilia may exist, but this is rarely seen, as attention may not be drawn 
to the condition sufficiently early. Associated with these polynuclear cells 
in the early stage we may find an increase in the number of endothelial cells. 
Eosinophile cells are frequently observed, but do not have any definite relation 
to tuberculosis as an infection. The red cells may be occasionally numerous, 
but are usually small in number. 1 

Secondary Tubercular Pleurisy. 

As a rule, tubercular pleurisy secondary to a pulmonary tuberculosis yields 
a liquid which is poor in cells, practically all of which are very much altered 
and in some cases very difficultly recognizable. The polynuclear types may 
predominate to such an extent that a distinct polynucleosis exists as evidence 
of a septic rather than a true tubercular pleurisy. The polynuclear cells are 
usually old, much deformed, and their nature recognizable only by staining 
their neutrophile granules. Where the infection is directly tubercular and 
not mixed (the latter, however, usually being the case), an approximately 
equal division of the polynuclear and mononuclear forms may obtain. In 
this type of pleurisy the eosinophile cells may be very numerous, in one case 
of Widal and Ravaut constituting 54 per cent, of the cellular elements. The 
endothelial cells may be, as in the primary tubercular pleurisy, sufficiently 
numerous to constitute a distinct endotheliosis. They are, however, single and 
very rarely grouped in masses. 

Pneumococcus Pleurisy. 

This is a truly septic type of pleurisy and is characterized by a distinct 
polynucleosis. In the early stages of this type of pleurisy, the endothelial 
cells may be very numerous, while in the later stages they may be much dimin- 
ished. In this, as in all types of pus accumulations, marked autolysis is present 
so that the cells may show extreme degeneration. As this condition progresses 
toward recovery, some of the polynuclears may be replaced by the mononu- 

1 See Bullock and Twichell (Am. Jour. Med. Sc, 1915, CXLIX, 848) for a study of exudates 
in artificial pneumothorax. 



PLATE XXXV. 




Exudate from Tubercular Pleurisy. (Eosin-hematoxylin Stain ) 



PLATE XXXVL 




Exudate in Pneumonic Pleurisy. (Eosin-hematoxylin Stain.) 



TRANSUDATES AND EXUDATES. 675 

clear lymphocytes; while if suppuration becomes extensive the polynuclears 
increase and autolysis becomes extremely marked. In such exudates the 
pneumococcus may be demonstrated by staining methods. 

Streptococcus Pleurisy. 

This type is especially associated with a polynucleosis. These cells are 
frequently observed undergoing karyolysis, the cell body usually being markedly 
degenerated. In its early stage it may be accompanied by an endotheliosis, 
but when well developed is usually associated with the presence of only a few 
isolated endothelial cells. Stained smears show streptococci in large numbers. 

Typhoid Pleurisy. 

In this type a lymphocytosis is usually observed along with an endothe- 
liosis, these latter cells being in large masses instead of in single isolated forms 
as observed in the secondary tubercular pleurisy. This point may be valuable 
in differentiating these two conditions, which are associated with an increase 
in the number of lymphocytes. The eosinophiles may be increased and 
red cells may be present in large numbers. The identification of the specific 
organism will serve as a positive differentiation from tubercular conditions. 

Malignant Pleurisy. 

This is a type of the aseptic pleurisies and may accompany malignant 
growths of the lung or pleura. Nothing characteristic is found in the cytologic 
formula of such exudates, but occasionally portions of the tumor mass may 
be obtained or certain cells may be found which are more or less distinctive, 
although not absolutely pathognomonic. These cells are frequently confused 
with the larger endothelial cells of the pleura, but are characterized, according 
to Deguy and Guillaumin, as follows: Malignant pleural cells contain glycogen 
which is recognized by the brown coloration shown on treatment with dilute 
iodin solutions, they contain large amounts of fatty material, they are ex- 
tensively vacuolated, these vacuoles may be large or so numerous and small 
that the cell resembles a sponge, the cells are extremely large, and the nucleus 
usually shows mitotic figures. 1 

Nephritic and Cardiac Pleurisy. 

The secondary exudate observed in cardiac and renal conditions is charac- 
terized especially by the marked endotheliosis. These endothelial cells are 
grouped in masses of 5 to 10 cells, which show more or less degeneration, their 
contours and their large nuclei being distinct. This endotheliosis is not com- 
plicated by the presence of many other cells in the pure nephritic pleurisy, 
but in the cardiac type we usually find a polynucleosis at the same time. This 
polynucleosis is much more marked where infarcts or emboli have complicated 
the condition than when it is due to a pure congestion. Numerous red cells 
are especially observed in association with a congestive pleurisy. 

Peritoneal Exudates. 

The cytological examination of peritoneal exudates has, as yet, yielded 

1 See Warren, Arch. Int. Med., 19 11, VIII, 648. 



676 DIAGNOSTIC METHODS. 

fewer diagnostic points than has that of pleural exudates. 1 It is, however, 
sometimes possible to differentiate a tubercular peritonitis from an ascites or 
an ovarian cyst by means of such examinations. Tubercular peritonitis 
usually shows a lymphocytosis and also a relative polynucleosis. A few 
endothelial cells may occasionally be found, but these do not yield much 
information. In ascites of hepatic origin few cellular elements are observed 
beyond the peritoneal endothelial cells. In ovarian cysts there are fewer 
cellular elements as a rule, but those present are usually large, round, or oval, 
and filled with vacuoles. Moreover, cylindrical ciliated epithelial cells as well 
as goblet cells or red cells are frequently relatively numerous. 

V. Cyst Fluids. 

(i). Ovarian Cysts. 
(a). Serous Cysts. 
Such cysts are true retention cysts formed by dilatation of the Graafian 
follicles and retention of the ovarian secretion and are known as Hydrops 
jolliculorum Graafii. They contain a clear, watery, serous liquid, which has 
an amber color, a specific gravity ranging between 1,005 an d 1,022, and a 
chemical composition practically identical with that of other serous fluids. 

(b). Myxoid or Colloid Cysts. 
These are proliferating cysts developed from the epithelial tubules. "We 
sometimes find in small cysts a semisolid, transparent, or somewhat cloudy 
or opalescent mass which appears like solidified glue or quivering jelly, and 
which has been called colloid because of its physical properties. In other 
cases the cysts contain a thick, tough mass which can be drawn out into long 
threads, and, as this mass in the different cysts is more or less diluted with 
serous liquids, their contents may have a variable consistency. In other 
cases the small cysts may also contain a thin watery fluid. The color of the 
contents is also variable. In certain cases it is bluish-white, opalescent, 
and in others yellow, yellowish-brown, or yellowish with a shade of green. 
They are often colored more or less chocolate-brown or reddish-brown, due 
to the decomposed blood pigment. The reaction is alkaline or nearly neutral. 
The specific gravity, which may vary considerably, is generally 1,015 to i >°3°j 
but may be, in a few cases, 1,005 to i>oio or 1,050 to 1,055. Though the 
contents of the proliferating cyst may have a variable composition, still it may 
be characterized, in typical cases, by its slimy or ropy consistency; by its 
grayish-yellow, chocolate-brown, sometimes whitish-gray color; and by its 
relatively high specific gravity. Such a liquid does not ordinarily show a 
spontaneous coagulation" (Hammarsten) . 

Microscopical examination of the sediment shows red and white blood- 
cells, large epithelial cells, which may be filled with vacuoles, cylindrical or 
goblet cells, granular cells showing more or less fatty degeneration, fatty granules, 

1 See Szecsi, Folia Haemat., 1912, XIII, 1. See Rolleston (Brit. Med. Jour., 1914, I, 238) 
and Weishaupt (Arch. f. Gynak., 1914, C, 496) for a discussion of eosinophilia in peritoneal 
exudates; also, Carslaw, Brit. Jour. Surg., 191 5, III, 1. 



PLATE XXXVII. 




Exudate in Malignant Pleurisy. (Eosin-hematoxylin Stain. 

Guillaumin.) 



After Deguy and 



TRANSUDATES AND EXUDATES. 677 

cholesterin crystals, and colloid corpuscles in the form of large, circular, highly 
refractile bodies. 1 

Chemically, these cysts are characterized by the presence of colloid, which 
is not a distinct chemical entity. It is a gelatinous substance, insoluble in 
water and acetic acid, soluble in alkalies, and yields a reducing body on boiling 
with acids. Not infrequently pseudomucin (metalbumin) is found, es- 
pecially in the extremely viscid fluids. For its detection the serum albumin 
must be previously removed by the addition of acetic acid, boiling, and filtering. 
The filtrate is treated with alcohol when a thready precipitate forms. If 
this precipitate be boiled with HC1, a substance is formed which reduces 
copper solutions quite markedly. Pseudomucin is distinguished from true 
mucin by the fact that it is not precipitated by acetic acid. Mitjukoff has 
isolated a further colloid body from certain ovarian cysts, to which he gives 
the name of paramucin. It is precipitated by acetic acid and is soluble in 
an excess. If treated with alkali it first swells up and then dissolves in an 
excess of the reagent. It differs from mucin and pseudomucin in the fact 
that it reduces copper solution without previous boiling with acids. McConnell 2 
has recently reported the finding of a multi-locular cyst of the ovary, which 
contained true mucin. 

(c). Papillary Cysts. 

These are intraligamentary types and contain a yellow, yellowish-green, 
or brownish-green fluid which contains only traces of pseudomucin. The 
specific gravity ranges between 1,032 and 1,036. 

(d). Dermoid Cysts. 
These may be seen in the shape of small cysts not larger than a pea, but 
usually they are much larger, in some cases reaching the size of a man's head. 
The cyst usually contains a fatty unctuous material, which is derived from 
the epidermal lining of the cyst, and associated with it fat, desquamated 
epithelial scales, hair, teeth, bone, cartilage, etc. 

(2). Parovarian Cysts. 

These cysts of the organ of Rosenmuller contain a clear, pale yellow, or col- 
orless, limpid fluid or occasionally one showing slight opalescence. The specific 
gravity ranges from 1,002 to 1,010 and differs from that of the ovarian cyst by 
its usual limitation to these lower figures. Albumin may be present in small 
amounts or be entirely absent, while pseudomucin is rarely, if ever, present. 

(3). Hydrocele. 

The contents of such a cyst are usually clear, show a color which may 
range from yellow to green, have a specific gravity of 1,015 to 1,030, and usually 
coagulate spontaneously. They contain a relatively large percentage of 
albumin, of which about 50 per cent, is globulin. In ordinary hydrocele many 
large oval cells may be seen, which have an eccentric nucleus and may be 
grouped in masses, although more frequently they appear as isolated cells; 

1 Dienst (Munch, med. Wchnschr., 1912, LIX, 2731) has called attention to the fact that 
fibrin is absent in the fluid from ovarian cysts while it is constantly present in ascitic fluids. 

2 Jour. Med. Research, 1909, XX, 105. ^_ 



678 DIAGNOSTIC METHODS. 

in some cases many eosinophils, but this is rare in uncomplicated hydroceles. 
If the hydrocele be of tubercular origin a marked lymphocytosis usually exists. 

(4). Spermatocele. 

Fluids from such cysts are usually thin, colorless, and cloudy like thin 
milk. They may have an acid reaction, but are ordinarily alkaline. The 
specific gravity ranges between 1,006 and 1,010. Such fluids do not coagulate 
either spontaneously or after the addition of blood. Microscopically, one 
observes cell detritus, fat granules, leucocytes, and spermatozoa. 

(5). Hydronephrosis. 

This is a true retention cyst of the kidney, due primarily to obstruction 
of the ureter, which may be either congenital or acquired. Material aspirated 
from a renal cyst is usually clear, but may be yellowish or reddish and distinctly 
turbid. Its specific gravity varies from 1,010 to 1,015, while the chemical 
composition is usually suggestive of urine. For some time the presence of 
urea and uric acid in hydronephrotic cysts was supposed to be pathognomonic, 
but it has been shown that these substances may be present both in ovarian 
and pancreatic cysts and may even be lacking in old renal cysts. Occasionally 
epithelial cells, derived from the uriniferous tubules may be found, but such 
cells are not always present nor are they sufficiently characteristic to be of 
great importance from the diagnostic standpoint. 

(6). Hydatid Cysts. 

The fluid obtained by puncture of an echinococcus cyst is usually clear 
and of an alkaline reaction, has a specific gravity varying between 1,005 and 
1,015, i s practically free from albumin, and contains a large amount of inorganic 
salts, especially of sodium chlorid. The characteristic findings of such a 
cyst are the hooklets, scolices, and shreds of faintly laminated membrane. 
In some cases no trace of any morphological elements can be found, but usually 
the diagnosis is rendered certain, especially if careful search be made, by the 
presence of some portion of the parasite or the cyst membrane. 

(7). Pancreatic Cysts. 

The puncture fluid from a pancreatic cyst varies in its physical properties, 
depending upon the nature of the cyst as well as the length of time the fluid 
has remained in the cyst. It is usually bloody in character, has a specific 
gravity ranging from 1,010 to 1,030 and may contain methemoglobin, hematin, 
and cholesterin. As characteristic constituents of such a cyst one finds fer- 
ments, which will digest all types of food material. 1 Such tests may be per- 
formed as outlined in the sections on Gastric Contents and Feces. 

VI. Cerebrospinal Fluid. 

Since the introduction of lumbar puncture by Quincke, the cerebrospinal 
fluid has gained more or less importance from the diagnostic point of view. 

1 See Christian (Arch. Int. Med., 1912, IX, 143) who reports peculiar findings in the fluid 
of an epigastric cyst; also Besley, Jour. Am. Med. Assn., 1914, LXII, ion; Ipsen, Hospital- 
stid., 1914, LVII, 889 and 921; Speese, Ann. Surg., 1914, LX, 673; Rosenthal, Arch. f.Ver- 
dauungskr., 1914, XX, 619; Eustachio, Rif. Med., 1915, XXXI, 424: Willis and Budd, Surg., 
Gyn. and Obs., 1915, XX, 688. 



TRANSUDATES AND EXUDATES. 679 

So much may be learned, either from the standpoint of direct or differential 
diagnosis, that every practitioner should be able to perform a lumbar puncture 
and to examine the fluid obtained. There is little danger in the procedure 
as the spinal cord does not reach to the point of puncture and the fibers of the 
cauda equina are sufficiently movable to escape the needle. While few bad 
effects are observed in the ordinary run of cases, a few have been reported 
in which symptoms of collapse were evident. It should be a rule, therefore, 
to stop proceedings if such symptoms arise and also to keep the patient quiet 
in bed for at least 24 hours following the puncture, so that the pressure in the 
cerebrospinal cavity may become equalized. 1 

Lumbar Puncture. 

The patient is placed upon his left side near the edge of the bed, the knees 
should be flexed upon the abdomen, and the site of puncture prepared as for 
any surgical procedure. The needle used for puncture should be from 5 to 
10 cm. long and have a lumen from 1 to 2 mm. in diameter. It is always wise 
to provide the larger needles with a stylet, so that tissue fragments or blood 
may not gain entrance accidentally to the tube and thus lead to possible 
diagnostic errors. This stylet may be removed the moment the needle pene- 
trates the dura mater. 

The site of puncture should be on a level with the junction of the third 
and fourth lumbar vertebrae at a point about 1 cm. to the side (preferably 
the upper) of the median line. The needle is directed slightly upward and 
inward, the depth to which the puncture should be made varying with the 
age of the patient, the younger the child the less the depth. This puncture 
should be made carefully and yet with sufficient force to penetrate easily 
the musculature. If any marked resistance arises, it is probable that the 
needle has struck the vertebra, in which case the pressure must be reduced or 
the needle may break. This is not an infrequent occurrence with those not 
used to the technic, so that it may be advisable for the student to practice the 
procedure upon the cadaver. As soon as the dural sac is reached, the cerebro- 
spinal fluid will flow from the canula, the rate of flow indicating in a general 
way the pressure of the fluid. No aspiration should be used at any time, 
as this procedure is extremely dangerous. 

It is frequently advisable to know exactly what this pressure is, so that 
one may resort to the following method as used by Sahli. As soon as the 
needle penetrates the dura, a connection is made with a mercury manometer 
by means of a rubber tube filled with a 1 per cent, solution of carbolic acid. 
The portion of the manometer above the level of the mercury, forming the con- 
nection between it and the carbolic acid tube, must also be filled with the 
fluid. The manometer is filled with mercury to the zero point and held in 
such a manner that this point is on a level with the point of the aspirating 

1 For discussions regarding the source of the spinal fluid and variations in it see Dixon and 
Halliburton, Jour. Physiol., 1913, XLVII, 215; Ibid., 1914, XLVTII, 129 and 317; Dandy and 
Blackfan, Jour. Am. Med. Assn., 1913, LXI, 2216; Frazier, Ibid., 1914, LXIII, 287; Cushing, 
Jour. Med. Research, 1914, XXXI, 1; Weed, Ibid., 21, 51 and 93; Wegefarth, Ibid., 119 and 
149; Wegefarth and Weed, Ibid., 167; Frazier and Peet, Am. Jour. Physiol., 1914, XXXV, 268; 
Weed and Cushing, Ibid., 1915, XXXVI, 77; Frazier, Jour. Am. Med. Assn., 1915, LXTV, 1119. 



68o 



DIAGNOSTIC METHODS. 



needle, which is possible with ordinary manometers only when the connecting 
tube is of considerable length. 1 Under normal conditions the dural pressure, 
in the dorsal position, ranges between 5 and 7.5 mm. of mercury, or 60 to 
100 mm. of water if a water manometer be used. In pathologic conditions, 
such as meningitis or brain tumor, it ranges between 15 and 60 mm. of 
mercury or 200 to 800 mm. of water. 

Normal cerebrospinal fluid is colorless, limpid, and free from morphologi- 
cal elements. Its specific gravity ranges between 1,002 and i,oio. 2 It is alka- 
line in reaction, the degree of alkalinity varying between 15 and 20. It contains 
a trace of protein and about 0.1 per cent, of glucose. The salt content of this 

fluid is, according to Zdarek, 0.836 
gram, of which 0.429 gram is referable 
to sodium oxid and 0.017 t0 potas- 
sium oxid. This would rather militate 
against the older statements that a 
large amount of potassium salts as 
compared with sodium salts was pres- 
ent. The relation of KC1 to NaCl is, 
according to Nawratzki, 1 to 18, while 
Zdarek gives this ratio as 1 to 40. 
The former figure agrees closely with 
that obtained by various workers with 
pathological cerebrospinal fluids. 3 
The amount of fluid obtained by 
lumbar puncture is extremely variable. 
In normal individuals this amount is 
unknown as puncture is rarely per- 

Fig. 159.— Lumbar puncture: a, Quincke's formed upon normal cases. Pathologi- 
site; b, Maran's site; c, Chipault's site. „ , , . , 

{Tyson.) cally, the amount obtained varies be- 

tween 10 and 100 c.c. Naturally, if 
the communication between the subarachnoid spaces of the brain and of the 
spinal cord is blocked by a tumor or inflammatory adhesions, or if the aque- 
duct of Sylvius or the foramen of Magendie be obliterated, little fluid may 
be obtained by puncture, although large amounts may be present above the 
obstruction. The largest amounts are seen in cases of serous or tubercular 
meningitis, so that such conditions may usually be ruled out if a small amount 
of fluid is obtainable. 

Pathologically, we may observe a very cloudy fluid, due to the presence 
of leucocytes, erythrocytes, and endothelial cells. This cellular admixture 
may be so extensive that the fluid resembles pure pus. In cases of cerebral 
hemorrhage from the ventricles, hemorrhagic pachymeningitis, or traumatic 
lesions of the spinal cord, so much blood may be present as to give the appear- 

t * See Strauss (Jour. Am. Med. Assn., 1914, LXII, 1327) for a special spinal-puncture needle 
with manometer attachment. 

2 See Stanford (Ztschr. f . physiol. Chem., 19 13, LXXXVI, 43 and 2 19), who shows that this 
density is higher in progressive paralysis and epilepsy than in other conditions. 
3 See Rosenbloom and Andrews, Arch. Int. Med., 1914, XIV, 536. 




TRANSUDATES AND EXUDATES. 68 1 

ance of practically pure blood, the color varying from a bright red to a brownish 
or greenish-red, depending upon the length of time it has remained in contact 
with the remaining portion of the fluid. This admixture with blood may 
lead to the spontaneous coagulation of the fluid. 1 This may serve as a differen- 
tiating point between inflammatory and noninflammatory lesions. Thus, 
in tubercular meningitis very slight coagulation may be observed, while in the 
epidemic cerebrospinal meningitis the coagulum may be very firm. 

The chemical examination of the cerebrospinal fluid offers some points of 
clinical value. While the albumin content normally is much less than o.i per 
cent., it may vary under pathologic conditions to as high as 0.8 per cent. The 
total protein, especially the euglobulin portion, is increased in all cases of acute 
exudative inflammations of the meninges, in hydrocephalus as Polanyi 2 has 
shown and, also, in syphilitic and parasyphilitic diseases of the cerebrospinal 
tract. Glucose is usually present but may entirely disappear under pathologic 
influences due to the autolysis controlled by the leucocytic ferments, the glucose 
being converted into lactic acid. 3 Cholin is present normally in traces, while 
pathologically it may vary, according to Donath, between 0.021 per cent, and 
0.046 per cent. 

Koch 4 has introduced the following method for examination of this fluid, 
which should extend our knowledge. The fluid is centrif uged and the sediment 
used for the bacteriological and cytological examinations. Ten c.c. of this 
clear fluid are placed in a beaker, 3 c.c. of a saturated solution of ammonium 
nitrate and 5 drops of nitric acid are added, and the mixture heated on a water- 
bath until complete coagulation has occurred. This precipitate of protein 5 is 
filtered through a perforated Gooch crucible, washed with 0.1 per cent, ammo- 
nium nitrate solution and then with alcohol, dried at ioo° C. and weighed. The 
filtrate is taken before the washing with alcohol is begun, concentrated to 10 to 
15 c.c. and 2 c.c. of 1 per cent, barium nitrate solution added while boiling. 
The barium sulphate precipitate is allowed to settle over night, is filtered off, 
dried and weighed. With the filtrate a phosphorus estimation is made by the 
molybdic method, with subsequent precipitation as magnesium ammonium 
phosphate in the usual way. Increase in the phosphorus and sulphur content 
is associated with degenerative changes in the nerve tissue. A test for reducing 
substances is made with 1 c.c. of the original fluid. 

1 Schwarz (Deutsch. Ztschr. f . Chir., 1913, CXXIV, 346) shows that a yellowish tint (xan- 
thochromasia) is frequently due to conversion of the blood pigment into urobilin and, hence, 
that this is a valuable indication of intracranial hemorrhage. See, also, Babes, Compt. rend. 
Soc. de biol., 1914, LXXVII, 67. For total nitrogen and urea of spinal fluid see Adler and 
Ragle, Boston Med. and Surg. Jour., 1014, CLXXI, 769; Woods, Arch. Int. Med., 1915, XVI, 
577; and Cullen and Ellis, Jour. Biol. Chem., 1915, XX, 511. Weston (Jour. Med. Research, 
1915, XXXIII, 119), discusses the cholesterol variations in this fluid. 

2 Biochem. Ztschr., 1911, XXXIV, 205. See, also, Ferrier, Lancet, 1913, II, 1107. 

3 See Kopetzky (Trans. Am. Acad, of Ophth. and Oto-Laryngol., 1912, 1, 261) who believes 
the absence of the copper-reducing body to be the earliest sign of bacterial invasion of the central 
nervous system. See Szabo, Ztschr. f. d. ges. Neurol, u. Psychiat., 1913, XVII, 145; Soper 
and Granat, Arch. Int. Med., 1914, XIII, 131. 

4 Arch. Neurol., 1907, III, 331; Arch. Neurol, and Psychiat., 1909, IV, 1. 

6 Mott advises the following method for protein: 10 c.c. of the centrif uged fluid are 
faintly acidified with acetic acid and 20 c.c. of absolute alcohol are added. The mixture is 
boiled and filtered, the precipitate of protein being dried and weighed. :J 



682 DIAGNOSTIC METHODS. 

Microscopic Examination. 

By far the most important part of the clinical examination of cerebro- 
spinal fluid is a study of the bacteriology and cytology of the fluid. Normally 
the fluid contains practically no morphologic elements, while under pathologic 
conditions large numbers of various types of cells may be present. The 
material for examination is obtained as previously described in the section 
on cytology. In true tubercular meningitis a lymphocytosis is almost invar- 
iably observed, while in the epidemic type, due to the meningococcus of Weich- 
selbaum, the cells are of the polynuclear type. In the chronic cases of epidemic 
meningitis as well as during convalescence from this disease, the lymphocytes 
may be present to such an extent that a slight degree of lymphocytosis is present, 
but never to the same extent as observed in the tubercular type. 1 A lymphocy- 
tosis is also observed in syphilitic lesions of the central nervous system. This 
is important from the standpoint of differential diagnosis. In certain cases of 
brain tumor, of non-specific origin, a lymphocytosis may obtain along with an 
increase in the protein-content of the fluid. In the meningitis due to the 
pneumococcus, a polynucleosis is the rule, although occasional cases are seen 
in which a lymphocytosis obtains. 2 In the prodromal stages and early days 
of acute anterior poliomyelitis a marked increase of fluid is accompanied by 
an increase in number of cells, which are largely of the mononuclear type. 
These are replaced by the polymorphonuclear forms as the number diminishes. 
In the microscopic examination of the cerebrospinal fluid it is sometimes of 
interest to know the number of cells per c. mm., of the fluid. The technic 
is as follows: The following mixture is employed for staining the white and dis- 
solving the red cells: Methyl violet 0.2 gram, glacial acetic acid 5 c.c, water to 
100. Fill the pipet with stain to the mark 1 and then fill to point n with un- 
centrifuged cerebrospinal fluid. Shake thoroughly, let stand for five minutes 
and count, using a Fuchs- Rosenthal counting chamber. The ruled surface of 
the cell contains 3.2 c. mm of fluid. Count all of the white cells in the entire 
ruled area, multiply by n and divide by 32. The result is the number of cells 
per c. mm., which varies from 1 to 10 normally. 3 

Epidemic Cerebrospinal Meningitis. 

In these cases the fluid may be transparent, but is usually somewhat 
opalescent and may be thick and purulent. The cellular elements are usually 
polynuclear in type and red cells may be more or less numerous. 

Smears made from the sediment show the presence of numerous diplococci, 
which closely resemble the gonococcus in morphological and staining character- 
istics. This organism, the diplococcus meningitidis intracellularis of Weichsel- 

1 Wassermann and Lange (Berl. klin. Wchnschr., 1914, LI, 527) believe that these lympho- 
cytes are, at least, one source of the substances responsible for the positive Wassermann reac- 
tions obtained with spinal fluid. 

2 See, however, Brady, Jour. Am. Med. Assn., 1913, LX, 972. 

3 See Blatteis and Lederer, Jour. Am. Med. Assn., 1913, LX, 811; Karpas, Ibid., 1913, 
LXI, 262; Engman, Buhman, Gorham and Davis, Ibid., 735; Brem, Ibid., 742; Maruyama, 
Wien. klin. Wchnschr., 19 13, XXVI, 1233; Ball, Interstate, Med. Jour., 1913, XX, 1109; 
Rubenstone, New York Med. Jour., 1913, XCVIII, 1210; Gordon, Ibid., 1914, XCIX, no; 
Mitchell, Darling and Newcomb, Jour. Nervous and Ment. Dis.; 1914, XLI, 686; Cottin, 
Rev. me*d. de la Suisse Rom., 1914, XXXIV, 715; Bigelow, Cleveland Med. Jour., 1915, XIV, 
196. 



TRANSUDATES AND EXUDATES. 683 

baum, appears as a diplococcus, each element forming a hemisphere with its 
parallel side contiguous to that of its mate. 1 It is sometimes seen in the form 
of tetrads or as isolated cocci, which appear as true spheres of variable size 
and showing a clear space in their interior. It is stained with the ordinary 
dyes and is negative to Gram's stain. For its cultural peculiarities see the 
last chapter of this book. Not infrequently one observes specimens of the 
meningococcus which show a Gram-positive reaction, so that it is difficult to 
distinguish them, especially when they are in the form of isolated cocci or in 
groups of two or four, from the pneumococcus. This type has been described 
as the meningococcus of Bonome, while Jaeger and Heubner describe a diplo- 
coccus which is Gram-positive and may be confused with the unusual types of 
Weichselbaum's meningococcus. 

Recently Flexner has succeeded in preparing an antimeningococcic serum 
which appears to have remarkable results in controlling this hitherto unmanage- 
able disease. In using this serum, the 
injection must be made directly into the 
spinal cavity. 2 More or less frequent in- 
jections of the serum and examinations of 
the lumbar fluid are made and the influence 
of the serum estimated by bacteriological 
and cytological examinations. In the cases 
of mixed cerebral infection, in which the 
meningococcus is associated with the 
pneumococcus, streptococcus, typhoid 
bacillus, staphylococcus, and other organ- 
isms, this serum does not seem to have as ^ ^. , . „ - . 
. . . Fig. 160. — Diplococcus lntracellulans 
much influence as in the pure meningo- meningitidis. (Councilman.) 

coccus infections. 3 

It is to be remembered that a purulent meningitis may be secondary 
to infection with practically all of the pus-forming organisms found within 
the system. It is, therefore, essential that any infection showing meningeal 
symptoms should be investigated by an examination of the cerebrospinal fluid. 

In sleeping sickness, a study of the cerebrospinal fluid frequently reveals 
the presence of the trypanosoma Gambiense. These parasites are not found 
in all cases, but when present usually furnish a grave prognosis. 

Recent investigation of the cerebrospinal fluid, applying Wassermann's 
serum reaction for syphilis, has shown that in the large majority of nervous 
cases of syphilitic origin a positive reaction is obtainable. 

1 See Frost, Pub. Health Rep., 1912, XXVII, 97; Broers and Smit, Nederl. Tijdschr. v. 
Geneesk., 1915, II, 1175; also, Hort, Lakin and Benians (Brit. Med. Jour., 1915, 1, 541), 
Foster (Ibid., 543) and Donaldson (Lancet, 1915, 1, 1333), who suggest that the meningococ- 
cus is only a phase in the life history of the true infective agent. 

2 See Dubois, Jour. Am. Med. Assn., 1913, LX, 820; also, Wollstein, Jour. Exper. Med., 
1914, XX, 201; Dubois and Neal, Am. Jour. Dis. Child., 191 5, IX, 1. 

3 Collignon and Pilod (Presse med., 1911, XIX, 732) advocate the following test to differ- 
entiate meningococcic from other types of meningitis. To a few c.c. (50 drops) of centrifuged 
spinal fluid add one to four drops of antimeningococcus serum and incubate the mixture (along 
with a control tube containing only spinal fluid) at 56 C. for 12 hours. If the serum and 
spinal fluid be homologous, a definite precipitation will be observed. This test appears to be 
specific. 







684 DIAGNOSTIC METHODS. 

Tubercular Meningitis. 

The fluid in such cases is usually clear, but may be slightly opalescent. 
The cellular elements are largely mononuclear while a few red cells may be 
present. If tubercle bacilli cannot be found after careful search, the presence 
of a lymphocytosis along with an increased protein-content will be at least sug- 
gestive, while animal inoculation or the tuberculin test will clear up the diag- 
nosis in most cases. (See Lucas 1 .) 
Acute Anterior Poliomyelitis. 

This disease has been definitely established as infectious through the work 
of Flexner and his associates. The causative agent was known to belong to 
the ultra- microscopic type and to pass the Berkefeld filter. In other words it 
is a filterable virus, the point of ingress and egress of which is the nasal and 
pharyngeal mucosa. This virus is transmitted by means of the biting stable-fly 
(stomoxys calcitrans) and probably, also, by the common fly and bedbug. 2 

Cultivation of this organism has been accomplished by Flexner and Noguchi. 3 
The culture medium consists essentially of sterile human ascitic fluid to which 
has been added a fragment of sterile fresh kidney tissue of the normal rabbit. 
Solid media is made by the addition of 2 per cent, agar to above. The exclu- 
sion of oxygen is necessary for the initial culture, but it suffices to cover the 
media with a deep layer of sterile paraffin oil. Films prepared from the lower 
layers of the fluid cultures are made, are air-dried and fixed in methyl alcohol 
for one hour. Wash in distilled water and immerse in Giemsa's solution (1 
drop of stain to 1 c.c. of distilled water) for two to twelve hours. Examination 
reveals minute globoid bodies arranged in pairs or short chains, or in small 
aggregated masses. These bodies are stained violet and average about 0.2 
micron in diameter. Their pathogenicity has been proven by inoculation of 
monkeys. 

The changes in the spinal fluid, while not absolutely diagnostic are sugges- 
tive. The amount of fluid usually increases early in the disease, this increase 
being associated with a relative increase in the mononuclear types. These 
cells may first be of the small lymphocytic variety but they soon change to the 
large mononuclear form. As the disease progresses, the cells diminish in num- 
ber, the polymorphonuclear forms assuming the ascendency. The total protein 
is increased in amount (as detected by the Noguchi or Ross- Jones method), while 
fibrin formation appears to be an early sign, although this may soon disappear. 4 

1 Am. Jour. Dis. Child., 1911, I, 230; also, Mandelbaum, Deutsch. Arch. f. klin. Med., 

1913, CXIII, 92; Roby, Jour. Am. Med. Assn., 1915, LXV, 1027. 

3 Rosenau, Discussion in 15th Cong, on Hyg. and Demog., Abs. in Jour. Am. Med. Assn., 
1912, LIX, 13 14; Anderson and Frost, Pub. Health Rep., 1912, XXVII, 1733; Howard and 
Clark, Jour. Exper. Med., 1912, XVI, 850; Mitzmain, Pub. Health Rep., 1913, XXVIII, 345; 
Lucas and Osgood, Jour. Am. Med. Assn., 1913, LX, 1611; Rosenau, Ibid., 1612; Sawyer and 
Herms, Ibid., 19 13, LXI, 461. 

3 Jour. Exper. Med., 1913, XVIII, 461. See, also, Amoss, Ibid., 1914, XLX, 212. 

4 See " A Clinical Study of Acute Poliomyelitis" by Peabody, Draper and Dochez, published 
by the Rockefeller Institute in 1912; Flexner, Science, 1912, XXXVI , 685; Fraser, Jour. Exper. 
Med., 1913, XVIII, 242; Kling and Levaditi, Ann. de l'lnst. Pasteur, 19 13, XXVII, 718; 
Cassel, Deutsch. med. Wchnschr., 1913, XXXIX, 2507; Lust and Rosenberg, Munch, med. 
Wchnschr., 1914, LXI, 121; Flexner and Amoss, Jour. Exper. Med., 1914, XIX, 411; Ibid., 

1914, XX, 249; Ibid., 1915, XXI, 509; Flexner, Noguchi and Amoss, Ibid., 91; Flexner, Bull. 
Johns Hopkins Hosp., 1915, XXVI, 180; Am. Jour. Dis. Child., 1915, IX, 353. See, also, 
Morse, Boston Med. and Surg. Jour., 1914, CLXX, 373; Corbus, Jour. Am. Med. Assn., 1914, 
LXIII, 550; Neisser, Berl. klin. Wchnschr., 1915, LII, 486. 



TRANSUDATES AND EXUDATES. 685 

Cerebrospinal Syphilis. 

Although such cases do not show pathognomonic findings in the cerebro- 
spinal fluid, yet certain factors are of importance in diagnosis. A lymphocyto- 
sis is usually observed together with a marked increase in the globulin content, 
which is estimated as follows: 1 

Noguchi's Butyric Acid Test. 

One part (0.1 or 0.2 c.c.) of spinal fluid is mixed with 5 parts (0.5 c.c.) of 
a 10 per cent, butyric acid solution in physiologic salt solution. This mixture 
is heated to boiling and immediately 1 part (0.1 c.c.) of normal (4 per cent.) 
sodium hydrate solution is added and the mixture again boiled for a few seconds. 
The presence of an increased content of protein in the fluid is indicated by the 
appearance of a granular or flocculent precipitate which gradually settles under 
a clear supernatant liquid. This precipitate appears within a few minutes in a 
specimen containing a considerable increase in protein, while two hours may be 
required to obtain a distinct reaction in specimens weaker in protein. May 2 
has called attention to the advisability of using larger portions of the fluid, 
preserving the same proportions of reagents as used by Noguchi. 

This reaction appears regularly in the cerebrospinal fluid of patients with 
syphilitic and parasyphilitic affections and also in all cases of inflammation of 
the meninges caused by such organisms as the meningococcus, pneumococcus, 
influenza bacillus, tubercle bacillus, etc. These latter cases are, however, 
easily differentiated from the syphilitic affections. Normal fluid gives a tur- 
bidity but the granular precipitate does not occur at all or only after many 
hours. 

Although this reaction is given in many non-syphilitic conditions, it has a 
certain value. It is not specific and, when present, does not necessarily indicate 
a syphilitic infection. On the other hand, it can be employed to establish or 
confirm a deduction based upon the clinical history and the results of the 
Wassermann reaction and cytodiagnosis, thus becoming of great indirect 
diagnostic value. A syphilitic infection is, however, practically excluded by a 
negative reaction. In this respect this test has advantages over the Wassermann 
reaction, in which a negative result is not always reliable as indicating absence 
of syphilitic infection. 3 

Nonne's Test. 

This test, 4 like the above, is for the detection of an increase of the protein 
material in the spinal fluid. It is, however, not limited to syphilitic conditions, 
although it is widely used for such cases. Nonne has advanced two steps which 
he styles "phases." 

1 See Kaplan and Casamajor, Arch. Int. Med., 1912, IX, 262; also, Ball, Jour. Am. Med. 
Assn., 1912, LIX, 1272. Ellis (Jour. Exper. Med., 1913, XVIII, 162) asserts that no syphilitic 
patient should be regarded as cured without a complete negative examination of the cerebro- 
spinal fluid. 

2 Arch. Int. Med., 1911, VIII, 183. 

3 Nichols and Hough (Jour. Am. Med. Assn., 1913, LX, 108) report the demonstration of 
spirochaeta pallida in the cerebrospinal fluid and the successful inoculation of a rabbit therefrom. 

4 Arch. f. Psychiat. u. Nervenkrankh., 1907, XLIII, 433; Munch, med. Wchnschr., 1907, 
LIV, 2 1 17. 



686 DIAGNOSTIC METHODS. 

Phase I. 

To a small portion of the clear cerebrospinal fluid add an equal volume of 
a hot saturated solution of ammonium sulphate. A trace of opalescence or a 
distinct turbidity, usually appearing within three minutes, indicates a positive 
reaction. Normal fluids never show any appreciable change with this test, 
while pathologic types always react promptly. The test is positive in all infec- 
tions of the nervous system and is of special interest in general paralysis, tabes 
and obscure cerebrospinal syphilis. Although not absolutely differential, it is, 
yet, of great importance, as a negative test excludes syphilis and parasyphilis. 

Phase II. 

In case no turbidity is observed in Phase I, add a drop or two of acetic acid 
to the above mixture. A distinct turbidity appears at once. This phase is 
given, not infrequently, with normal fluids so that little value is attached to it. 

Ross -Jones Test. 

This x is a modification of Phase I of Nonne and is, in my opinion, somewhat 
more delicate. Two c.c. of a saturated solution of ammonium sulphate are placed 
in a test-tube and i c.c. of the cerebrospinal fluid is gently run on to the surface 
so as to form a contact layer. A clear-cut, thin, grayish-white ring is observed 
at the junction of the liquids if the reaction be positive. This ring should ap- 
pear within three minutes. The value of the test is the same as that above. 

Lange's Colloidal-gold Test. 

As neither the Nonne nor Ross- Jones tests exclude inflammatory processes 
in the brain or cord, a more differential test is to be desired. This seems to 
have been found in Lange's test. It is based on the fact that protein material 
precipitates colloidal gold at a dilution which is constant for definite proteins. 
If the protein be in less concentration a protective power over the colloidal 
particles is exerted and no precipitation occurs. In the case of spinal fluid, 
the protein present in different pathologic conditions shows a different precipi- 
tating point in syphilitic and non-syphilitic disorders. This test is, therefore, 
much more differential than are the preceding ones given. 

Preparation of the Colloid Solution. 
Place 500 c.c. of double-distilled water in a Jena flask of 1,000 cc. capacity 
and heat slowly. When the temperature is about 6o° C, add, while still heat- 
ing, 5 c.c. of 1 per cent, solution of gold chlorid (Merck's C. P.) and follow 
immediately with 5 c.c. of 2 per cent, solution of potassium carbonate. Heat 
rapidly to boiling and discontinue heating. Add quickly 5 c.c. of 1 per cent, 
dilution of formalin (not of formaldehyd). All these solutions must be added 
with a pipet as the amounts must be accurate in order to insure success in the 
preparation of the fluid. Shake the flask vigorously until a change of color 
occurs, beginning with a darkening of the fluid and passing through faint blue, 
dark blue, and purple to a final red. This is the finished colloidal solution and 
should be a distinct magenta and absolutely clear. Preserve in a dark-colored 
bottle. 

1 Brit. Med. Jour., 1909, I, 1111. 



TRANSUDATES AND EXUDATES. 687 

Technic. 

Place in a rack a series of twelve test-tubes. In the first, place with a pipet 
1.8 c.c. of 0.4 per cent, freshly made sodium chlorid solution. In the other 
eleven tubes place 1 c.c. of the chlorid solution. To the first tube add 0.2 c.c. 
of spinal fluid, which must be free from blood. The dilution in tube 1 is, there- 
fore, 1 to 10. Mix this thoroughly and place 1 c.c. of this first dilution in the 
second tube. This dilution is 1 to 20. Proceed in the same way with the 
remaining tubes, getting dilutions up to 1 to 20,440. 

After the dilutions are prepared, add to each tube 5 c.c. of the colloidal 
solution and let the tubes stand for 24 hours at room temperature. The read- 
ing of the test is as follows: The original tube showed a medium magenta 
shade. As the precipitation of the gold occurs, the color of the tubes con- 
stantly manifests a bluer tinge and finally becomes clear and colorless. The 
dilution at which the greatest precipitation occurs is noted and the results at 
various dilutions expressed by the + or — sign. Thus — indicates no precipi- 
tation; + , a red with beginning blue tinge; ++, red-blue and blue-red 
shades; + + + , violet and dark blue colorations; + + + + , light blue color; 
+ + + + + , complete precipitation with a clear and colorless solution. 1 

In syphilitic and parasyphilitic cases the tendency of the precipitation is 
constantly toward the left of the series, that is toward the 1 to 10 dilution. 
The maximum effect in such conditions is 1 to 40 or 1 to 80. It never continues 
past 1 to 640 and infrequently past 1 to 160. In non-sylphilitic cases the tend- 
ency is toward the right of the series, that is toward the 1 to 20,000 dilution, the 
maximum being observed at some point above 1 to 80. Tuberculous menin- 
gitis shows the most intense reaction at 1 to 160 or 1 to 320, while suppurative 
meningitis gives most marked reactions at 1 to 1,200 and up. This reaction 
promises to be of great value, especially in congenital syphilis. 

BIBLIOGRAPHY. 

1. Adami. Principles of Pathology. Philadelphia, 1908. 

2. Kraus und Levaditi. Handbuch der Immunitatsforschung. Jena, 1908. 

3. Jordan. General Bacteriology. Philadelphia, 1908. 

4. Mestrezat. Le Liquide Cephalo-Rachidien. Paris, 19 12. 

5. Oppenheimer. Handbuch der Biochemie. Jena, 1908. 

6. Plant, Rehm, and Schottmuller, Leitfaden zur Untersuchung der Zere- 

brospinalflussigkeit. Jena, 19 13. 

1 Berl. klin. Wchnschr., 1912, XLIX, 897; Ztschr. f. Chemotherap., 1912, I, 44. See, also 
Grulee and Moody, Jour. Am. Med. Assn., 1913, LXI, 13; Eicke, Munch, med. Wchnschr., 
1913, LX, 2713; Kaplan and McClelland, Jour. Am. Med. Assn., 1914, LXII, 511; Miller and 
Levy, Bull. Johns Hopkins Hosp., 1914, XXV, 133; Kafka and Rautenberg, Ztschr. f. d. ges. 
Neurol, u. Psychiat., 1914, XXII, 353; Lee and Hinton, Am. Jour. Med. Sc, 1914, CXLVIIT, ^y, 
Debenedetti, Rif. Med., 1914XXX, 906; Brock, 111. Med. Jour., 1914, XXVI, 422; de Crinis and 
Frank, Munch, med. Wchnschr., 1914, LXI, 1216; Kafka, Ibid., 1915, LXII, 105; Solomon and 
Koefod, Boston Med. and Surg. Jour., 1914, CLXXI, 886; Solomon and Welles, Ibid., 1915, 
CLXXII, 398 and 625; Weston, Darling and Newcomb, Am. Jour. Insan., 1915, LXXI, 835; 
Grulee and Moody, Am. Jour. Dis. Child., 1915, IX, 17; Barnes and Ives, Interstate Med. 
Jour., 1915, XXII, 792; Swalm and Mann, New York Med. Jour., 1915, CI, 719; Rubenstone 
and Schwartz, Ibid., 1273; Busquet, Bull. l'Acad. Med., 1915, LXXIV, 183. 



CHAPTER X. 
SECRETION OF THE MAMMARY GLANDS. 

I. General Considerations. 

The normal secretion of milk takes place in the mammary glands of the 
female after delivery. It is true that a small quantity of milk may be secreted 
by the new-born of both sexes for a few days after birth. Moreover, cases 
have been reported in which the adult male secreted sufficient milk to act 
as a wet-nurse, but these must be regarded as cases of extreme rarity. 

During the course of a normal pregnancy a small amount of a thin, yellowish 
fluid may be expressed from the mammary glands, but as a rule the first real 
secretion is observed following delivery of the child. This secretion is thin 
and watery, more or less translucent, and shows a distinct yellowish color. 



i o ° o . 



• 2, ° c ©.» 






j o 



"*'v^ O 






'_ 



,y 






Fig. i 6 i. —Normal Milk and Colostrum. (Hawk.) 
a, Normal milk; b, colostrum. 

Microscopic examination shows the presence of rather large cells in which 
are many fat granules and occasionally a distinct nucleus. This secretion, 
which is called colostrum, 1 continues for three to four days and is distinguishable 
from the later secretion by the fact that it contains relatively more salts than 
normal milk and, according to the usual statements, more sugar. In a series 
of determinations of breast milk collected during the fourth day after delivery, 
the writer could not show any marked increase in the sugar in all cases, but 
in three out of eight cases examined a percentage higher than eight of lactose 
was obtained. 

1 See Engel and Bauer, Die Biochemie und Biologie des Kolostrums, Wiesbaden, 1912, 
Bergmann; Benestad, Monatsschr. f. Geburtsh. u. Gynak., 1914, XL, 674. 

688 



SECRETION OF THE MAMMARY GLANDS. 689 

It is probable that the function of the colostrum is gradually to accustom 
the child to the taking of food by diminishing to a slight extent the elements, 
protein and fat, which are more apt to cause digestive disturbances than 
is the lactose. 

The secretion of true milk begins about the fourth day and continues 
for a variable length of time. Marked variations are observed both in the 
quantity and the quality of this secretion in various women so that no hard 
and fast rule can be given as to the composition of normal mother's milk. 
While it is probably true that the normal woman should nurse her child during 
a large part of the first year, it is rare to find, especially in private practice, 
many such cases Either the milk becomes scanty and loses in nutritive power 
or becomes excessive and consequently diluted. In either case the child is 
not receiving the most suitable nourishment, so that breast-feeding is abandoned 
under these circumstances. It seems to be a general rule, which is difficultly 
explicable, that the more highly socially developed the woman the less apt 
she is to nurse her child with any success. It is possible, and this seems to 
the writer the most probable explanation, that the child usually receives more 
or less constant attention from the physician, and variations in the breast 
milk are more frequently noticed than in the case of the poorer child who 
rarely has the advantage of medical attention unless more or less serious illness 
occurs. 

II. Physical and Chemical Properties. 

Various figures have been given for the composition of human milk so 
that it is difficult to strike an average. So much depends upon the nourishment 
of the mother, upon the amount of exercise taken by her, and upon the general 
condition of the system that marked variations exist in the proportion of the 
chemical constituents. The following table, taking averages of examinations 
reported by various writers, may serve as one representing more nearly the 
normal condition. 1 



Water, 


87.24 


Salts, 


0.26 


Protein, 


1.50 


Fat, 


4.00 


Lactose, 


7.00 



These figures are not as high as regards protein as those usually given, 
but are higher as far as the lactose is concerned. Although many writers 
have given the percentage of protein much higher than 1.5 per cent., the writer 
has never been able, in several hundred examinations of milk in his laboratory, 
to find many showing a protein percentage of two or more. 

As it is frequently necessary to modify cow's milk so that it may more 

1 See Schloss, Monatsschr. f. Kinderhkde., 1912, X, 499; also, Meigs and Marsh, Jour. 
Biol. Chem., 1913, XVI, 147; Talbot, Am. Jour. Dis. Child., 1914, VII, 445; Grulee and Cald- 
well Ibid., 1915, IX, 374; Hart and Humphrey, Jour. Biol. Chem., 1915, XXI, 239. 

44 



690 DIAGNOSTIC METHODS. 

nearly approach mother's milk in composition, the writer inserts the following 
table for comparison. 



Water, 


87.25 


Salts, 


0-75 


Protein, 


3-5o 


Fat, 


4.00 


Lactose, 


4-5° 



It will be seen that cow's milk shows a higher per cent, of protein and a 
lower per cent, of carbohydrate. It is necessary, therefore, that this be 
modified by diluting the milk so as to diminish the protein and by adding 
lactose to make up for the deficiency of carbohydrate. In the dilution the 
fat content will necessarily be lowered so that this may be remedied, as sug- 
gested by Backhaus, by the addition of cream. The writer must refer to 
works on pediatrics for the various methods of modifying cow's milk. It is 
to be remembered that no modification is equal to mother's milk. The casein 
of human milk forms a much finer clot with the gastric juice than does that 
of cow's milk so that the latter may not be well tolerated by the child. More- 
over, some unknown principle present in human milk is accountable for a 
distinct biologic difference in these two types of nutritive material. 

(1). Appearance and Color. 

Normal human milk is a white fluid which usually has a slight bluish 
tinge except immediately after birth when the color may be distinctly yellowish 
from the presence of colostrum. If the percentage of fat be relatively high 
the color will be more nearly a pure white with little of the blue tone. 

(2). Specific Gravity. 

The specific gravity of human milk varies between 1,028 and 1,034. An 
increase in the percentage of fat will usually lower the specific gravity while 
the protein and carbohydrates will increase it. 

Cow's milk shows approximately the same specific gravity. If a low 
specific gravity is obtained it is evidence either of an increased percentage 
of fat or of dilution with water. If the percentage of fat be low the milk 
is unquestionably a watered one. If the milk tested shows a high specific 
gravity the chances are that most of the fat has been removed either by skim- 
ming or by centrifugation. 

The determination of the specific gravity may be made by a special in- 
strument known as Quevenne's lactodensimeter or by the use of the ordinary 
hydrometer used in urine work. As it is never of any clinical importance 
or even of any marked practical importance that the specific gravity should 
be absolutely accurately determined, the writer is accustomed to use the 
urinometer for such determinations. Corrections for variations in tempera- 
ture are necessary only when great differences exist between the tempera- 
ure of the room and the temperature at which the instrument is calibrated. 



SECRETION OF THE MAMMARY GLANDS. 69 1 

(3). Reaction. 

Normal human milk as well as cow's milk shows an amphoteric reaction 
to litmus-paper and an acid reaction to phenolphthalein, cow's milk being 
somewhat more acid than mother's milk toward the latter indicator. 1 

(4). Coagulation. 

If milk be allowed to stand, the reaction gradually becomes more and 
more acid owing to the development of bacteria, especially of the bacillus 
acidi lactici. When the degree of acidity reaches a certain point, casein sepa- 
rates first in the form of flocculi and later the entire fluid may coagulate to 
a jelly-like mass. This mass soon contracts and settles out leaving a slightly 
turbid fluid known as milk plasma or acid whey. 2 In order to inhibit the 
development of bacteria and prevent this coagulation, known as souring, 
certain preservatives are frequently added to market milk and should be 
capable of detection by the practitioner. These will be discussed in a later 
section. 

Besides this type of coagulation of cow's milk a second form is observed 
which takes place under the influence of chymosin without any change in the 
reaction of the fluid. In this case the whey is sweet and contains practically 
all of the lactose originally present in the milk. 

(5). Total Solids. 

Five to ten c.c. of the well-mixed milk are placed in a weighed platinum 
dish, evaporated to dryness on a water-bath, and dried to constant weight 
in the oven at 105 . The difference -in weight between the platinum dish 
and its contents, on the one hand, and the platinum dish, on the other, gives the 
amount of total solids in the milk taken. A simple calculation will yield the 
percentage of total solids. 

Under normal conditions the total solids of both human and cow's milk 
should average between 12 and 13 per cent. Variations in this figure are, 
of course, due to fluctuations in the various constituent elements. 

(6). Ash. 

The platinum dish containing the dried residue of the milk is heated 
over a direct flame until the residue is completely incinerated. This is then 
placed in the desiccator and dried to constant weight. The difference in 
weight between the dish and contents and the dish alone represents the amount 
of salts present in the milk originally taken. 3 

(7). Protein. 

The methods for the determination ot protein are divided into those for 
estimation of the total protein material present and into those in which separate 
determinations are made of the casein, on the one hand, and albumin and 
globulin, on the other. 

1 See Clark, Jour. Med. Research, 1914, XXXI, 431; Milroy, Biochem. Jour., 1915, IX, 215. 

2 See Orla- Jensen, Ztschr. f. physiol. Chem., 1914, XCIII, 283. 

3 Bosworth, Jour. Biol. Chem., 1915, XX, 707; Holt, Courtney and Fales, Am. Jour. Dis. 
Child., 1915, X, 229. 



692 DIAGNOSTIC METHODS. 

(a). Total Protein. 
Method of Sebelien. 

Ten c.c. of milk are diluted with 90 c.c. of water, 5 c.c. of a saturated so- 
dium chlorid solution, and 15 c.c. of Almen's tannic acid solution are added. 
The mixture is thoroughly stirred and the dense precipitate which forms is 
allowed to settle. The composition of Almen's tannic acid solution is as follows: 

Tannic acid, 4 grams. 

Acetic acid (25 per cent.), 8 c.c. 

Alcohol (50 per cent.), qs., ad., 200 c.c. 

The precipitate, which consists of the total protein of the milk and a large 
portion of the fat carried down by the precipitate, is then filtered off through 
a fine filter and is washed with cold water. The filter-paper and its contents 
are then placed in a Kjeldahl flask and a nitrogen determination made as 
described under Urine. It is advisable to use 20 c.c. of sulphuric acid instead 
of the ten employed in the case of urine, as the mixture oxidizes much more 
rapidly under these conditions. If the nitrogen obtained in this determination 
be multiplied by 6.37, the result will be the protein in 10 c.c. of milk. 

Method of Boggs. 

For routine work this method is perhaps more advisable for the general prac- 
titioner than is the preceding, but it does not always give confirmatory results. 
It is based upon the fact that the total protein of milk is precipitated by phospho- 
tungstic acid in hydrochloric acid solution, the amount of precipitate being 
measured in an Esbach tube. The reagent used has the following composition; 

Phosphotungstic acid, 25 grams. 

Concentrated hydrochloric acid, 25 c.c. 

Distilled water, q. s., ad., 250 c.c. 

It has been found that the milk should be diluted with water before adding 
the reagent if the results are to be accurate. As a rule, a dilution of 1 to 10 
for human milk and 1 to 20 for cow's milk suffices. 

The diluted milk is poured into the Esbach tube 1 to the mark U and 
the reagent added to the mark R. The tube is then closed with a stopper 
and inverted several times thoroughly to mix the contents. It is then set 
aside for 24 hours when the percentage of protein in the milk is read off directly 
from the calibrations on the tube in case the dilution was 1 to 10, while with 
a dilution of 1 to 20 the figures are multiplied by 2. 

This method, while convenient, is open to the objection that many factors 
may influence the depth to which a precipitate settles. Moreover, the Esbach 
tubes reading as high as 12 parts do not give as satisfactory results as those 
with readings from one to seven. Such being the case the method must be 
used more for clinical purposes than for scientific estimations. 

1 If a Purdy centrifuge tube be filled with the diluted milk to the 10 c.c. mark and the 
reagent added to 15 c.c, the amount of protein may be determined quickly by centrifuging 
for 3 minutes and reading the percentage directly. The figures obtained agree quite closely 
with those of the gravimetric method. f ? 



SECRETION OF THE MAMMARY GLANDS. 693 

(b). Casein. 

Twenty c.c. of well- mixed milk are measured into a beaker and approxi- 
mately 380 c.c. of water are added. The mixture is thoroughly stirred and 
very dilute acetic acid added drop by drop with constant stirring until a flocculent 
precipitate is observed. When this point is reached a stream of carbon dioxid 
is passed through the mixture for one-half hour, after which the vessel is 
allowed to stand until the next day. The above part of the technic is directly 
applicable to cow's milk. If human milk is being examined it is necessary 
to heat the vessel to 40 C. during the passage of the carbon dioxid. 

After the mixture has stood overnight, it is filtered through a nitrogen- 
free filter and washed with water. The residue on the filter contains casein 
which is mixed with a portion of the fat present. The filter-paper and contents 
are then placed in a Kjeldahl flask and a nitrogen determination is made as 
previously described. Multiplication of the nitrogen value by 6.37 yields 
the amount of casein in the 20 c.c. of milk originally taken. 1 

(c). Albumin and Globulin. 

The filtrate from the above precipitation of casein contains the remainder 
of the protein material and the carbohydrate of the milk. This filtrate is placed 
in a porcelain dish and heated for a few minutes to the boiling-point. The 
protein material is coagulated and may be filtered through a nitrogen-free 
filter and washed several times with cold water. A nitrogen determination 
is then made and the value multiplied by 6.37 to obtain the amount of albumin 
and globulin present in the 20 c.c. of milk. 

The filtrate from this latter precipitation contains the lactose, which may 
then be determined by titration with Fehling's solution, as described under 
Urine. It is to be remembered that 10 c.c. of Fehling's solution are reduced 
by 0.0678 gram of lactose, and not by 0.05 as in the case of glucose. 

(8). Fat. 

It is important in the determination of the fat content of milk that a 
thoroughly mixed specimen be examined. 2 As the fat tends to rise to the 
surface of the milk, the fluid should be poured from one vessel into another 
several times to insure thorough mixing and an immediate measurement 
made of the portion to be tested. 

For clinical purposes as well as for examination of market milk the method 
of Babcock is to be recommended. For accurate results, however, this method 
is not to be advised. 

Babcock's Method. 

This method consists in the destruction of the organic matter, except 
the fat, by means of sulphuric acid. The fat is then separated by centrifuging 
and determined by reading off the percentage from the calibrations in the 
neck of the bottle used. 

In the case of cow's milk or where sufficient human milk may be obtained, 

1 See Van Slyke and Bosworth, Jour. Biol. Chem., 19 15, XX, 135. 

2 See Brodrick-Pittard, Biochem. Ztschr., 1914, LXVII, 382; Osborne and Wakeman, Jour. 
Biol. Chem., 1915, XXI, 539. 



694 



DIAGNOSTIC METHODS. 



17.6 c.c. of milk are measured into the bottle and 17.5 c.c. of sulphuric acid 
added. These fluids are then mixed by shaking and rotating the bottle in 
such a way that no curds pass into the neck of the bottle. As soon as the 
mixture becomes homogeneous and dark brown or even black in color, the 
bottles are placed in a special centrifugal machine and whirled for five minutes. 
If the room be very cold it is advisable to fill the holders of the centrifuge with 
boiling water in order to keep the fat melted while it is being centrifuged. 
At the end of five minutes, centrifugation is discontinued and the neck of the 
bottle filled with boiling water. The melted fat will rise 
in the neck of the flask and may be read off from the cali- 
bration. In order to facilitate this the bottles are again 
centrifuged for one minute. 

If a small amount of milk only is available the smaller 
tubes shown in cut may be used. Milk is added to the 
mark five and sulphuric acid poured in so as to fill the body 
of the tube. It is usually necessary to add the milk and 
acid by means of thin narrow pipets, as the neck of the 
tube is too small to permit of easy entrance of the fluids 

otherwise. The milk and sul- 
phuric acid are mixed by rotation 
of the tube until a homogeneous 
fluid results. The mixture is 
centrifuged for a few minutes, 
the neck of the tube being filled 
with a mixture consisting of 
equal parts of concentrated 
hydrochloric acid and amyl 
alcohol. The percentage of fat 
is then read off from the calibra- 
tions on the tube. 



>CC 



Fig. 162. — Babcock Bottles. 
a, Milk bottle; &, cream bottle. 



Fig. 163.— Bottle 
for human milk. 



Extraction Method. 

A few grams of dried washed 
sand are placed in the extraction 
shell of a Soxhlet apparatus and 
10 c.c. of well-mixed milk are allowed to fall upon it drop by drop. This is 
dried at a temperature of ioo° C. for one to two hours and is placed in the 
tube of the extraction apparatus. The fat is extracted in the usual way by 
the use of gasoline or anhydrous ether, complete extraction usually requiring 
from two to three hours. The apparatus is disconnected, the ether evapo- 
rated from the distilling flask, the residue in the flask dried at ioo° C, and 
the flask and contents dried to constant weight in the desiccator. The differ- 
ence between the original weight of the flask and its weight including the ex- 
tracted residue, yields the amount of fat in the 10 c.c. of milk. This method 
is the most accurate one, but is not as convenient as the preceding for the 
general practitioner. 



SECRETION OF THE MAMMARY GLANDS. 



695 



(9). Lactose. 

In general routine analyses of milk the lactose may be determined by 
difference. By this is meant that subtraction of the values for the sum of 
water, ash, protein, and fat from 100 will yield 
the percentage of lactose. For clinical purposes 
this is usually sufficient, but for the more ac- 
curate work it does not give exact figures, as 
slight amounts of other undetermined sub- 
stances are present. 

For any direct determination of lactose it 
is necessary that the larger portion of the 
protein material be removed previously. This 
may be done by the method outlined under 
Determination of Casein, Albumin, and 
Globulin; or for clinical purposes sufficiently 
accurate results may be reached by acidifying 
with acetic acid, boiling, and filtering. It is 
advisable always to take the time to saturate 
the mixture with carbon dioxid after the 
casein has been precipitated with acetic acid, 
as the results are more satisfactory. 1 

The fluid is then titrated by the use of 
Fehling's or Purdy's solution, using all the 
precautions mentioned under these tests in the 
section on Urine. Ten c.c. of Fehling's solu- 
tion are reduced by 0.0678 gram of lactose, 
while the 35 c.c. of Purdy's solution are re- 
duced by 0.02712 gram of lactose. 

If the polarimeter is to be used for the 
estimation of lactose in milk the following 
procedure may be used. Fifty c.c. of the well- 
mixed milk are placed in a flask, 25 c.c. of a 
solution of neutral lead acetate are added, the flask is closed with a stopper 
through which passes a glass tube approximately 30 cm. in length. The 
mixture is then heated over a small flame to boiling. After the mixture has 
cooled it is filtered through a dry filter into a dry vessel and polarized. 

(10). Preservatives in Cow's Milk. 
(a). Sodium Carbonate. 

To hide the acid reaction of a spoiled sample of milk sodium carbonate 
is often added, and may be detected as follows: Ten c.c. of milk are mixed 
with ten c.c. of 96 per cent, alcohol and a drop of rosolic acid solution. Pure 
unadulterated milk produces a brownish-yellow color, but, in the presence 
of sodium carbonate or bicarbonate, a rose color is obtained. For greater 

1 See Jackson and Rothera, Biochem. Jour., 1914, VIII, 1; Palmegiani, Pediatria, 1914, 
XXII, 739; Hill, Jour. Biol. Chem., 1915, XX, 175. 




Fig. 164. — Soxhlet apparatus. 
(Hawk.) 



696 DIAGNOSTIC METHODS. 

precision in doubtful cases the questionable sample should be compared with 
known unadulterated milk. Phenol-phthalein solution may be used as an in- 
dicator in place of rosolic acid. By this method 0.05 per cent, of carbonates 
may easily be detected. 

(b). Salicylic Acid. 
Twenty c.c. of milk are treated with two or three drops of sulphuric acid 
and shaken with an equal amount of ether. The greatest possible part of the 
ethereal solution is drawn off and evaporated, the residue extracted with 
40 per cent, alcohol, filtered, and 5 c.c. of the filtrate treated with a few drops 
of ferric chlorid solution. A violet color shows the presence of salicylic 
acid or some other hydroxy derivative of benzol. 

(c). Formaldehyd. 
Two c.c. of concentrated sulphuric acid are placed in a test-tube and a 
drop of ferric chlorid solution added. A few c.c. of milk are allowed to run 
from a pipet upon the surface of the mixture in such a way that a distinct 
line of contact forms. A violet color at the point of contact of the two liquids 
is characteristic of formaldehyd in the presence of casein. 

(d). Boric Acid and Borax. 

Fifty c.c. of milk are alkalinized with milk of lime, evaporated to dryness 
and incinerated. The resulting white crystalline residue is treated with a 
few drops of tincture of turmeric and very dilute hydrochloric acid and is 
then dried on the water bath. The presence of the slightest trace of boric 
acid gives to the dry residue a beautiful vermilion or cherry red color. It is 
possible by this method to detect 0.001 per cent, of boric acid in milk. Only 
very dilute hydrochloric acid must be used in testing for boric acid, since the 
concentrated acid itself gives with tincture of turmeric a red color. The 
coloration produced by boric acid is distinguished from that produced by 
hydrochloric acid by the fact that it does not disappear by treatment with 
water in the cold, but only after long boiling, while the color caused by hydro- 
chloric acid disappears as soon as it is diluted with water. 

If the crystalline residue obtained as above described be treated with 
alcohol and the alcohol ignited, a flame tinged with a beautiful emerald-green 
color is obtained in the presence of boric acid. 

III. Bacteriological Examination of Milk. 

The bacteriological examination of human milk is frequently desirable 
from a . clinical standpoint as milk may become contaminated as it passes 
along the lacteal ducts. In pathologic conditions many types of organisms, 
such as typhoid bacilli, pneumococci, and tubercle bacilli may be obtained, 
although the latter are extremely rare. 

With cow's milk the chief question at issue is whether the milk contains 
sufficient bacteria to be harmful to the child. Aside from the presence of the 



SECRETION OF THE MAMMARY GLANDS. 697 

tubercle bacillus in the milk of infected animals, large numbers of saprophytic 
organisms must find their way into this fluid and will, if in large numbers, 
influence the intestinal activity of the child. Moreover, it must be remembered 
that many epidemics of milk-borne disease, such as typhoid fever and strepto- 
coccic sore-throat, may arise owing to improper handling of the milk or to 
inefficient pasteurization. 1 

The methods of examining milk for bacteriologic differentiation are the 
same as for any other fluid and will be discussed in the next chapter. It is, 
not infrequently, a matter of importance to the general practitioner and of 
untold value to health officers to determine the number of bacteria in the milk 
supplied to the community. The outline of the method to be followed in this 
work is as follows: 

Collect the specimen as freshly as possible. Keep it on ice from the time 
of collection until you are ready for the examination. Sterilize a quantity ^of 
distilled water in the autoclave. Plug a number of pipets (1 and 10 c.c.) with 
cotton and sterilize in the hot oven. A number of Erlenmeyer flasks, plugged 
with cotton, a 100 c.c. graduate, and a number of Petri dishes are sterilized at 
the same time. 




Fig. 165. — Wolfhugel's colony counter. 

By means of the graduate place 99 c.c. of sterile distilled water in an Erlen- 
meyer flask, 90 c.c. in a second and 99 c.c. in a third and replace the cotton. 
By means of a sterile 1 c.c. pipet transfer 1 c.c. of the well-shaken specimen 
to flask number 1. The dilution of the milk in this first flask is, therefore, 1 
to 100. Shake this dilution thoroughly and transfer 10 c.c. of this dilution, by 
means of a pipet, to the second flask containing 90 c.c. of water. The second 
dilution is 1 to 1000. By means of a second 1 c.c. pipet transfer 1 c.c. of the 1 to 
100 dilution to the last flask, containing 99 c.c. of water, thus obtaining a dilu- 
tion of 1 to 10,000. These dilutions are usually all that are necessary. Others 
may be made as desired. In the examination of the best market milks, the 
1 to 100 dilution is sufficient. 

Liquefy a number of tubes of litmus-lactose agar, litmus-lactose-bile agar 
and plain agar and cool them down to 40 to 45 C. Transfer 1 c.c. of each 
dilution of the milk to each of two sterile Petri dishes and add the liquefied 
culture medium. Mix by careful manipulation and allow the media to harden, 
Place in incubator for 48 hours and count the number of colonies, using a 

1 See discussion of " Streptococcic Sore Throat" on page 40; also Rogers and Dahlberg, 
Jour. Agricul. Research, 1914, I, 491. Shippen (Jour. Am. Med. Assn., 1915, LXTV, 1289) 
shows, in conformation of the work of De Jong and De Graef, that colon bacilli may survive 
the pasteurization process and are, therefore, not to be taken as evidence of later contamination. 



698 DIAGNOSTIC METHODS. 

colony counter such as shown in the illustration. Multiply the number of 
colonies by the dilution to obtain the actual number of colonies per c.c. of original 
milk. 1 

The number of bacteria in milk may vary from a few thousand to many 
millions. It is an impossibility to obtain a specimen which is sterile so that 
the presence of as high as 100,000 organisms should not be regarded as danger- 
ous unless pathogenic types are present. If the freshly examined specimen 
contains between 10,000 and 50,000 bacteria per c.c, it is probable that all 
possible precautions have been taken to prevent contamination, although the 
writer has found some specimens showing a count of only 100 per c.c. 

In the examination of milk it is customary also to make a cytological 
examination. It was formerly supposed that normal fresh milk contained only 
a few scattered leucocytes and epithelial cells, any marked increase in their 
number being taken as evidence of infection. However, by use of more careful 
methods it has been shown that the number of these cells may run as high as 
500,000 or more and yet no abnormality be present. It is necessary in this 
work to distinguish between the ordinary leucocytes, which are constantly dis- 
charged in large numbers in the milk, and the true pus cells, which are evidence 
of disease. No detailed work along this line has been reported. The associa- 
tion of large numbers of bacteria, especially of staphylococci and streptococci, is 
probably evidence of infection of the animal and consequent unfitness of the milk 
for use, although the significance of the increase in cellular elements is not, by 
any means, settled. The methods used in this cytological examination are the 
same as outlined previously. 2 

BIBLIOGRAPHY. 

1. Czerny und Keller. Die Nahrungs-Pathologie. Berlin, 1907. 

2. Holt. Diseases of Infancy and Childhood. New York, 1906. 

3. Langstein and Meyer. Sauglingsernahrung und Sauglingsstoffwechsel. 

Wiesbaden, 19 14. 

1 See Brew, New York Agricultural Exper. Station, Geneva, N. Y., Bull. 373, 1914; also, 
Goodrich, Jour. Infect. Dis., 1914, XIV, 512. 

2 See Lewis, Am. Jour. Dis. Child., 1913, VI, 225; Hachtel, Jour. Am. Med. Assn., 1913, 
XI, 565; Stokes and Stoner, Ibid., 1024; Breed, Bull. 380, New York Agricultural Exp. Station, 
Geneva, N. Y., 1914; Hewlett and Revis, Lancet, 1915, I, 855; Frost, Jour. Am. Med. Assn., 
191 5, LXTV, 821. 



CHAPTER XL 
CLINICAL BACTERIOLOGY. 

I. General Considerations. 

In no field of medical or other scientific research have there been recent 
advances of such far-reaching importance to humanity as in the realm of 
Clinical Bacteriology. The study of the relationship of bacteria and their 
metabolic products to disease in general and. the identification of the specific 
organism of many of our most dreaded diseases has led to such an enormous 
output of work upon the various ways in which the system may react to bac- 
terial invasion, that our knowledge of the subject of immunity, both natural and 
acquired as well as active and passive, has advanced with leaps and bounds. 
Indeed, so greatly has our knowledge increased that preventive medicine is 



Fig. 166. — Lautenschlager Hot-air Sterilizer. 

fast taking the place of curative medicine. It is evident, therefore, that the 
scientific aspect of bacteriology, great as it is, is, for the general worker and for 
the public at large, overshadowed by the success attached to the practical ap- 
plication of the knowledge acquired from the hard and earnest work of scores 
of scientific investigators. Scientific and applied bacteriology must go hand 
in hand, each playing its important part in the struggle against disease. 

The object of the writer in presenting this section is not to include a dis- 
cussion of the advances in bacteriology, as this would, indeed, be absurd. He 
does wish, however, to present certain general features of the subject, so that the 

699 



700 



DIAGNOSTIC METHODS. 



student and practitioner may be given such a working knowledge as will enable 
them properly to perform and interpret many of the tests, which they may 
have occasion to undertake. Many such points have been discussed throughout 
this book but many have been omitted, so that the writer feels that this dis- 
cussion will not be untimely. 



II. Sterilization. 




As is well known, bacteria are practically 
ubiquitous, being found in air, water, food and 
soil. It is evident, therefore, that all material 
and glass-ware, which may be used in bacterio- 
logical work, must be clean and sterile before 
any reliable results may be obtained. 

Glass-ware should be thoroughly cleaned 
by boiling in soapsuds or by soaking for a 
few hours in the following cleaning mixture. 



60 grams. 
300 c.c. 
460 c.c. 



Potassium bichromate, 

Water, 

Concentrated sulphuric acid, 

After washing or soaking, rinse the glass 
thoroughly in running water and dry. Plug 
the test-tubes and flasks with non-absorbent 
cotton and place in a hot-air sterilizer or oven 
for one hour at a temperature of 170 C. 
The internal temperature of the sterilizer must 
be carefully regulated or the variation may be 
so great in different portions of the sterilizer 
that the more resistant organisms and spores 
will not be killed. 

Culture media are best sterilized by the use 
of the autoclave, an apparatus for holding steam 
under pressure. The usual pressure used is 15 
pounds, the media being kept in contact with 
steam at this pressure (temperature about 
120 C.) for 15 to 20 minutes. The gas is then 
turned off, and the apparatus allowed to cool. While most media may be 
sterilized by this method, gelatin media would better not be heated longer 
than five minutes and blood-serum and carbohydrate-containing media may 
undergo chemical change if the heating is prolonged. 

For this reason, such media as last mentioned or all media, in the absence 
of an autoclave, may be sterilized in the Arnold sterilizer, an apparatus so con- 
structed as to produce steam from a small amount of water, the tank being 
fed by the water of condensation. As many bacterial spores are not killed by 
live steam, it is necessary to use the so-called discontinuous sterilization when 



Fig. 167. 



CLINICAL BACTERIOLOGY. 



7OI 



thejArnold is employed. Heat the media for 30 minutes after the steam has tilled 
the sterilizer and allow the media to stand at room temperature for 24 hours. 
Repeat the process on each of two other successive days. In this way spores 
develop into vegetative organisms, which are killed by the later heating. 

Certain fluids, such as serum or ascitic fluid, cannot be sterilized by heating 
owing to coagulation. These are sterilized by passing through a Pasteur, 
Chamberland or Berkefeld filter. 




Fig. 168. — Arnold Sterilizer. 



III. Preparation of Culture Media. 



For the development of bacteria certain factors are essential. The most 
important of these are (1) oxygen; (2) food; (3) moisture; (4) a proper reaction 
of the culture media; and (5) an optimum temperature for growth. 

Regarding the question of proper food it is to be said that the number of 
culture media advocated is legion. I cannot, therefore, attempt to give any 
but those in general use in laboratory routine. Many special media have been 
discussed in other sections of this book and will be referred to later in the 
discussion of special organisms. 

Nutrient Broth (Bouillon; Beef Broth). 

Infuse 500 grams of chopped lean meat with 1,000 c.c. of distilled water 
for 24 hours in the ice box. Instead of this meat, one may use 3 grams of 
Liebig's meat extract, but the medium is not as nutritious. Strain the infusion 
through cotton flannel, the juice being well pressed out. Add 1 per cent, pep- 
tone (10 grams). Warm on water bath, stirring until the peptone is dissolved. 
Heat over boiling water or steam for 30 minutes. Restore the original volume 



702 



DIAGNOSTIC METHODS. 



of fluid. Titrate the acidity of the broth, using N/20 sodium hydrate solution 
and phenolphthalein as an indicator. 1 Adjust the reaction to + 1 by adding 
the calculated amount of normal sodium hydrate. Boil two minutes over free 
flame, stirring constantly. Restore loss by evaporation and filter through paper 
or absorbent cotton. Titrate and accurately adjust the final reaction to + 1. 
Place the broth in test-tubes, using 10 c.c. in each tube. Plug tubes with cotton 
and sterilize in the autoclave. 

Sugar-free Broth. 

Make the infusion of meat (do not use meat extract) as above. Add to 
this 10 to 20 c.c. of a 24-hour broth culture of bacillus coli communis and incu- 
bate at 37 C. for 18 hours. Boil the mixture to kill the organisms and proceed 
as above, using this material as if it were ordinary meat infusion. 

Sugar Broths. 

Add to the sugar-free broth above mentioned 1 per cent, of dextrose, lactose 
or other sugar. Adjust the reaction of these broths so that it is neutral to 
phenolphthalein. Sterilize by the discontinuous method. 2 

Nutrient Gelatin. 

To the nutrient broth add 10 per cent, of " gold label " sheet gelatin. Warm 
on water bath, stirring till gelatin is dissolved but not allowing the temperature 
to rise above 6o° C. Heat over steam bath for 30 minutes and restore loss by 
evaporation. Titrate, after boiling one minute to expel carbon dioxid, and 
adjust the reaction to +1 as above described. Boil two minutes over free 
flame, stirring constantly. Make up loss by evaporation and filter through 
paper or absorbent cotton. If a clear filtrate be not obtained, it may be assured 
by cooling the mixture to 6o° C. and stirring in the white of an egg beaten up 
in 30 c.c. of water. Titrate and adjust final reaction. Tube as above and 
sterilize in the autoclave at 120 C. for five minutes. On being removed from 
the autoclave the gelatin is placed at once in the ice-box. 

Nutrient Agar. 3 

Prepare the meat infusion as given under nutrient broth. Weigh the filtered 
infusion and add 2 per cent, of Witte's peptone. Warm on water bath stirring 
until the peptone is dissolved and not allowing the temperature to go above 6o° C. 
While this mixture is being prepared, boil 15 grams of thread agar in 500 c.c. 

1 The optimum reaction for such culture medium is one having a neutral reaction to litmus 
and a degree of acidity requiring 1 c.c. of normal (4 per cent.) sodium hydrate to neutralize 
100 c.c. of the medium. This is known as + 1. See Clark, Jour. Infect. Dis., 19 15, XVII, 109. 

Place 5 c.c. of the medium to be tested in a porcelain dish and add 45 c.c. of distilled water. 
Boil to expel carbon-dioxid. Add 1 c.c. of 1/2 per cent, solution of phenolphthalein in 50 
per cent, alcohol. Titrate, while hot, with N/20 sodium hydrate up to a faint but distinct 
pink color. Read from the buret the amount of N/20 NaOH used to neutralize the 5 c.c of 
medium and calculate the amount of normal NaOH necessary to neutralize the entire quantity 
of bouillon. 

Thus, if 1.5 c.c. of N/20 NaOH were used for 5 c.c, this would represent 30 c.c for 100 
or 300 for 1000 c.c. But 300 c.c of N/20 NaOH is equivalent to 15 c.c of normal NaOH. 
As we desire the medium to have an end-reaction of + 1 per hundred or 10 per liter, we must 
evidently add 5 c.c of normal NaOH to bring this medium up to the proper standard. 

2 See Holman, Jour. Infect. Dis., 1914, XV, 209; also Vedder, Ibid., 1915, XVI, 385. 

8 See Report of Committee on Standard Methods of Water Analysis, Jour. Infect. Dis., 
1907, Supplement III, 108; also, Huber, 111. Med. Jour., 1915, XXVIII, 207. 



CLINICAL BACTERIOLOGY. 703 

of water for one-half hour and make up the weight to 500 grams. Let it cool 
to 6o° C. To 500 grams of the meat-peptone infusion add 500 grams of the 
3 per cent, agar, keeping the temperature below 6o° C. Heat over steam bath 
for 30 minutes and restore loss by evaporation. Titrate and proceed as given 
above for nutrient gelatin. Great difficulty is sometimes experienced in filter- 
ing this preparation, but if the filter paper and funnel be wet with boiling water, 
the medium usually passes through easily. Sterilize in the autoclave. 

Glycerin Agar. 

Prepare nutrient agar in the usual way. After filtering add 6 to 8 per cent, 
of glycerin. Mix thoroughly, readjust the titer if necessary and place in test- 
tubes. Sterilize in autoclave. This medium is preferable to plain agar in cul- 
tivating such organisms as the tubercle bacillus, which does not grow at all well 
on plain agar. 

Lactose and Dextrose Litmus Agar. 

To nutrient agar, prepared from sugar-free broth instead of nutrient broth, 
1 per cent, of lactose or dextrose may be added just before sterilization, which 
must be in the Arnold sterilizer. Adjust the reaction to the neutral point to 
phenolphthalein. If the medium is to be used in tubes, the sterilized litmus 
solution or preferably azolitmin solution shall not be added until just before the 
final sterilization. If it is to be used in Petri dishes, the sterilized solutions 
shall not be added until the medium is ready to pour into the dishes. The 
litmus solution used is a 1 per cent, solution of Merck's purified litmus extract. 
Of this 5 or 6 per cent, is added. It is, probably, preferable to use instead of 
this, a 1 per cent, solution of Kahlbaum's azolitmin. 

' < Ameba ,, Agar. 

This is the medium used by Musgrave and Clegg in cultivating the ameba 
and differs from nutrient agar in its strength. It is prepared in the same way 
but has the following formula 

Extract of beef, o. 3 to o. 5 gram. 

Sodium chlorid, 0.3 to o. 5 gram. 

Agar, 20.0 grams. 

Water, 1 000.0 c.c. 

The reaction is made — 1, that is, it is 1 per cent, alkaline. Variations maybe 
made in this medium, where the very delicate organisms are concerned, by 
adding a very small amount of peptone. 

Blood Agar. 

Prepare about 40 tubes of nutrient agar and, while still unsolidified, place 
them in a slanting position so that a large surface is given. Such tubes are 
called slants. Place 50 c.c. of nutrient agar in a flask and melt it. Allow it to 
cool to about 45 C. and add 15 c.c. of human blood, which has been drawn by 
venous puncture. Mix the contents of the flask by shaking and pour 2 c.c. of 
this mixture over the surface of each agar slant. Place these slants in posi- 
tion again so that the added material may harden. These tubes cannot be 
resterilized. 



704 DIAGNOSTIC METHODS. 

Instead of this method, which is preferable, one may add 1 to 2 c.c. of the 
human blood directly to 10 c.c. of agar melted and cooled to 40 or'45 C. Mix 
by shaking and slant the tubes. This medium is especially valuable for routine 
work as it will grow practically all organisms. 

Ascitic or Hydrocele Agar. 

Dissolve 15 grams of agar in 1,000 c.c. of water. Filter and place 5 c.c. in 
each test-tube. Sterilize in the autoclave. To each agar tube add 5 c.c. of 
sterile ascitic or hydrocele fluid and mix by rotating the tube. Cool so as to 
form slants. This medium cannot be resterilized. It is not always the sim- 
plest procedure to prepare this medium as the body fluid may be contaminated. 

Litmus Milk. 

Fresh milk, as pure as possible, is obtained and steamed for fifteen minutes 
in the Arnold. It is then placed in the ice-box over night to allow the cream to 
separate. The cream is drawn off and the skimmed milk used for the medium. 
The reaction should be adjusted to + 1. Add 1 per cent, azolitmin solution 
and tube 10 c.c. in test-tubes and sterilize for five minutes in the autoclave or 
in the Arnold for three successive days. 

Blood-serum. 

This is the medium devised by Loffler for the cultivation of the diphtheria 
organism and is very frequently used for others as well. It is prepared by add- 
ing 1 part of neutral nutrient broth containing 1 per cent, dextrose to 3 parts of 
sterile beef-serum. The mixture is then mixed thoroughly and placed in cul- 
ture boxes or, preferably, in short tubes. In the latter it should be slanted. 
Coagulate at 75 C. After being held at this temperature until coagulated, 
the serum may be very slowly heated to about 95 C. 

The above are the more important culture media used in routine work. 
Special media will be mentioned in discussing specific organisms. 

IV. Incubation. 

Although bacteria may develop at temperatures above that of 37 C. (body 
temperature) or at much lower temperatures, yet the optimum temperature for 
most bacteria is 37 C. It is true that many organisms develop more luxuri- 
antly and more rapidly at 20 C, but these bacteria are, for the most part, 
non-pathogenic. 

It is essential, therefore, that some sort of apparatus be at hand, which 
will permit of the maintenance of a regular and uniform temperature of 37 C. 
Such an arrangement is called an incubator, many types of which may be 
obtained. Some of these are heated by gas and others by electricity, the latter 
being by far the more preferable. A heat-regulator is attached so that variations 
in the temperature inside the incubator may be reduced to a minimum. In these 
incubators must be placed a dish of water to supply the necessary moisture, the 
development of bacteria proceeding the better the more nearly saturated the 
atmosphere of the incubator. 

Cultures when made upon the various media are placed in the incubator 



CLINICAL BACTERIOLOGY. 705 

for periods ranging from 24 hours to 10 days or more depending on the organism 
under investigation. As a rule 24 or 48 hours is the usual period of incuba- 
tion. Owing to the rapidity of liquefaction of the medium, gelatin-containing 
media cannot be placed in a temperature of 37 C. Such a medium must be 
used at room temperature (about 20 C). 



Fig. 169. — Incubator. 

V. Preparation of Cultures. 

As previously stated, oxygen is absolutely essential for the development of 
all bacteria. Some organisms are capable of growth in free oxygen (atmos- 
pheric air), while others cannot live in such an atmosphere but obtain their 
oxygen from the culture media upon or in which they develop. The former 
bacteria are known as aerobic organisms, while the latter types are called an- 

45 



yo6 DIAGNOSTIC METHODS. 

aerobic bacteria. The majority of the pathogenic organisms belong in the 
aerobic division, although many important pathogens are anaerobic. 

A. Aerobic Organisms. 

In this group we rind the diphtheria bacillus, tubercle bacillus, influenza 
bacillus, typhoid bacillus, cholera spirillum, gonococcus, meningococcus, 
pneumococcus, and many others. 

By means of the platinum needle, which is absolutely indispensable in bac- 
teriological work, the material to be investigated is spread over the surface of 
the proper culture medium (in routine work agar or blood agar) and the cotton 
plug replaced. These plugs do not prevent access of air but keep out dust and 
contaminating bacteria. Place in the incubator and allow the culture to de- 
velop for 24 or 48 hours. Instead of the medium in the tubes, one may use 
liquefied agar or blood agar. Place the liquid material under investigation 
(such as milk or water) in a sterilized Petri dish and pour in the liquefied medium, 
which has cooled down to 40 or 4 5 C. 



Fig. 170. — Platinum Needles. 

Study the macroscopic characteristics of the culture and prepare smears 
from the culture for microscopic examination. If the cultures are pure, that 
is if only one type of organism is present, sub-cultures may be made upon special 
media in order absolutely to identify the organism. If several types are present, 
each kind of colony is transferred to other media and isolated in pure culture 
and then identified by sub-cultures. Where simple diagnosis is desired, these 
sub-cultures are rarely necessary, but where a pure culture is at issue, as in the 
preparation of a vaccine, very careful differentiation is essential. 

B. Anaerobic Organisms. 

In this class one finds the tetanus bacillus, Treponema pallidum, Vincent's 
spirillum, organism of anterior poliomyelitis and many others. 

Cultures are made as in the case of aerobic organisms. However, these 
anaerobic types must develop in complete absence of free oxygen. To insure 
the absence of oxygen four methods are available. 

(1) Boiling the culture media. Allow the medium to cool down to 40 to 
45° C. and inoculate. This method is not at all reliable, owing to the possible 
access of air after boiling. 

(2) Displacement of the oxygen of the air by an inert gas, usually by hy- 
drogen. Inoculate the culture medium as above. Place in a larger vessel 
provided with entrance and exit tubes and tightly sealed. Pass a stream of 
purified hydrogen through the vessel continuously for several hours, close the 
stoppers and place the apparatus in the incubator. 



CLINICAL BACTERIOLOGY. 



707 



(3) Absorption of the oxygen. This is the more commonly employed 
method. Place the culture tubes on a glass or metal support inside a larger 
tube or bottle. Place in the bottom of the bottle 10 grams of pyrogallol and 
pour on it 100 c.c. of 1 per cent, sodium hydrate solution. Tightly stopper 
the bottle and place in the incubator. 1 

(4) The use of a vacuum. This method consists in exhausting, with an 
air pump, the air in a large vessel containing the inoculated culture media. 
Close the tubes to prevent reentrance of air and place in the incubator. After 
the air is exhausted, hydrogen may be passed into the vessel, thus giving double 
assurance of freedom from oxygen. 




— a 



Fig. 171. — Anaerobic Cultivation in Hydrogen Gas (Heinemann) . a, Novy jar; b, glass 
cock; c, gas generator; d, sulphuric acid; e, sodium hydrate solution;/, opening of Novy jar; 
g, stopper. 

After the cultures have developed they are studied and examined as men- 
tioned above. Sub-cultures and isolation of the different types are not so fre- 
quently essential with these anaerobic organisms, as the contamination is 
usually due to aerobic bacteria, which do not, of course, develop in these 
cultures. 



VI. Staining. 

The general methods of staining bacteria depend upon the special organism 
under investigation. As a rule Loffler's methylene blue (see p 21) and Gram's 
stain (see p. 668) are the ones which give all the general information de- 
sired. Certain organisms, however, require special staining, which methods 
will be discussed under the specific organism. Organisms are divided into two 
groups according to their reaction to Gram's stain, so that the writer would 
advise the constant use of this stain in order properly to classify an organism 
as positive or negative to this stain. 

Staining of Spores. 

Certain organisms show within their protoplasm a small bright refractile 
spot, known as a spore. These are very resistant and develop into vegetative 

1 The following method is very successful for routine work. Push the cotton plug into the 
tube a short distance, place dry pyrogallic acid on the upper portion of the cotton and pour on 
it 5 drops of 10 per cent, sodium hydrate solution. Stopper tightly with cork and seal it in 
with paraffin. 



708 DIAGNOSTIC METHODS. 

organisms very rapidly. The spore is not stained readily, but when once stained 
is very resistant to decolorization, the body of the bacillus losing its color before 
the spore does. In ordinary stained preparations the spore shows as an un- 
stained spot in the body of the stained bacillus. Of course, the spore may be 
stained with the simple stain, if the action be prolonged or if the stain be heated, 
but this does not give clear differentiation. 1 

Moller's Method. 

Make a smear of the culture. Dry in air and fix in absolute alcohol for two 
minutes followed by chloroform for two minutes. Dry. Drop a few drops 
of 5 per cent, aqueous solution of chromic acid on the smear and leave for two 
minutes. Wash in water. Cover the smear with Ziehl's carbol fuchsin (p. 
20) and warm for one minute. Wash in water and decolorize in 5 per cent, 
sulphuric acid for a few seconds, completing the decolorization, if necessary, 
with absolute alcohol. Wash in water. Stain with methylene blue for one-half 
minute. Wash in water, dry and examine. The spores are stained red and the 
body of the organism is blue. 

Staining of Capsules. 

Many methods have been advocated for this purpose, but the writer has 
found the method of Rosenow (see p. 590) the most satisfactory, giving beautiful 
pictures and uniform results. 

Staining of Flagella. 
Van Ermengem's Method. 

Place the film for five minutes at 50 C. (or 30 minutes at room temperature) 
in the following freshly prepared solution. 

20 per cent, aqueous solution of tannic acid, 60 c.c. 
2 per cent, solution of osmic acid, 30 c.c. 

Glacial acetic acid, 4 or 5 drops. 

Wash in water and then in absolute alcohol. Cover the smear with 1/2 per cent, 
aqueous solution of silver nitrate for a few seconds and, without washing, trans- 
fer the slide to the reducing bath for a few seconds. This reducing bath is as 
follows: 

Gallic acid, 5 grams 

Tannic acid, 3 grams 

Fused sodium acetate, 10 grams 

Distilled water, 350 c.c. 

Without washing, place the slide in the 1/2 per cent, silver nitrate solution and 
move the liquid back and forth over the smear until this becomes black. Wash 
in water, dry and examine. 

Successful preparations by this method, or any other, depend upon the 
obtaining of thin specimens. Take a small amount of a young agar culture of 
the organism and make a homogeneous emulsion in a watch glass with dis- 

1 See Huntoon, Jour. Am. Med. Assn., 1914, LXII, 1397. 



CLINICAL BACTERIOLOGY. 709 

tilled water. Place a drop or two of this emulsion on a perfectly clean glass 
slide and allow the drop to spread out. Dry in air and stain as above. 

VII. Identification of Organisms. 

After cultures have been prepared as above described and have been incu- 
bated for 24 to 48 hours, the diagnosis of a given infection may be possible by 
direct examination of the cultures and stained slides made therefrom. 1 This 
is true in cases in which search is being made for a specific organism without 
any reference to the associated bacteria. However, in the closer study of cul- 
tures, which is required in many cases, especially in the preparation of autog- 
enous vaccines, sub-cultures on special media must be made in order properly 
to purify the culture and to permit of differentiation and recognition of the organ- 
ism causing the infection. For this reason the writer deems it advisable to 
discuss briefly the cultural characteristics on special media and the means of 
absolute identification of some of the more important pathogenic bacteria. 2 

1. Diphtheria Bacillus (Klebs-Loffler Bacillus). 

The medium best adapted for the development of the diphtheria bacillus 
is Lomer's blood serum. 3 By means of a stout platinum needle or a sterile 
swab remove a portion of the suspicious material or membrane from the nose, 
throat or tonsils and spread this over the surface of the above culture medium. 
If the material is to be sent to a laboratory and no culture medium be at hand 
place the swab in a sterile test-tube or homeopathic vial and plug this with 
cotton. It is very essential that the proper material be obtained for these 
cultures or otherwise negative results will obtain. If necessary tear away the 
membrane in order to be certain of your material. 

Place the culture in the incubator at 3 7 C. for six to eight hours and examine. 
The cultures should rarely be examined after more than 20 hours to insure 
the best results. The growth appears as a mass of grayish-white points or colo- 
nies varying in size from a small to large pin-head. They are somewhat more 
opaque in their center than at their margin, which may be tinged a light yellow. 
The longer the period of growth, the larger the colonies, the more irregular their 
borders (early cultures usually show a regular margin) and the greater the 
tendency for the colonies to show radial striations. These colonies on blood 
serum always remain discrete and do not flow together. 

Smears made from such cultures are stained with methylene blue and with 
Neisser's or Gram's stain as given on page 39. Such smears show the organ- 
isms with the characteristic morphology and staining peculiarities given there. 

1 It is important to remember that transmutations may appear within the groups of bacteria 
so that pneumococci, for instance, may change into true streptococci by variations in the 
culture medium, by changing the oxygen tension within the culture tube, etc. See Thiele and 
Embleton, Ztschr. f. Immunitatsforsch. und exper. Therap., 1913, XIX, 643; Rosenow, Jour. 
Infect. Dis., 1914, XIV, 1; Jordan, Proc. Nat. Acad. Sc., 1915, I, 160. 

2 For a differential method of study of various bacteria by the addition of various aniline dyes 
to the culture medium see Churchman, Jour. Exper. Med., 1912, XVI, 221; Ibid., 1913, XVII, 
373; Churchman and Michael, Ibid., 1912, XVI, 822; Krumwiede and Pratt, Ibid., 1914, XLX, 
20 and 501; Simon and Wood, Am. Jour. Med. Sc, 1914, CXLVII, 524. 

3 See Drigalski and Bierast (Deutsch. med. Wchnschr., 19 13, XXXIX, 1237) who advise 
the addition of ox bile to this medium. 



710 DIAGNOSTIC METHODS. 

It is to be remembered that such cultures and smears may show no diphtheria 
bacilli, although the case is clinically one of diphtheria. This may be due to 
the taking of improper material for the culture, to the fact that antiseptic solu- 
tions had been used prior to the time of taking the culture or to the fact that too 
long a period of incubation has been used. Under the latter condition other 
organisms will so far outgrow the diphtheria bacilli that the latter are unrecog- 
nizable. For these reasons it must be insisted upon that a negative bacteriologic 
examination should never be regarded as excluding diphtheria. 

In some very urgent cases recourse is made to the examination of direct 
smears from the swab without preliminary cultivation. This method rarely, 
if ever, gives results which may be relied upon but it occasionally does. It 
would seem to the writer that cultures should be insisted upon in all cases, as 
the time necessary for obtaining a growth is so slight that one is warranted in 
waiting for a more definite diagnosis. All suspicious throats, especially in 
children, should be regarded as diphtheritic until proven to be otherwise by 
several cultures. This precaution is best both for the patient and for his 
associates. See Schick's immunity test (p. 605). 

It not infrequently happens that certain organisms, such as Hofmann's 
bacillus (pseudo-diphtheria bacillus) and the xerosis bacillus, complicate the 
diagnosis, as these organisms resemble the diphtheria bacillus in many ways. 
Cultures of Hofmann's bacillus, grown on LorHer's medium, show smaller and 
whiter colonies than those of the diphtheria bacillus. The growth of the former 
is much more rapid and there is not the same tendency for the colonies to take 
up the pigment of the medium and become yellowish in color. If Hofmann's 
bacillus be grown for 48 hours in glucose broth to which litmus has been added, 
no acid is produced; while the diphtheria bacillus readily produces acid (red 
coloration of medium). This fact has been mentioned on page 40, whereon 
are given the different sugars upon which these conflicting organisms act with 
production of acid but no gas formation. This fermentative test is of great 
value in differentiation. 

Further, the morphology of Hofmann's bacillus is not the same as that of 
the Klebs-Loffler bacillus, although at times the two may become confusing. 
The former is shorter than the latter (although the latter occasionally shows a 
short form); the former is oval, stains deeply throughout except for one un- 
stained portion and shows no granules. Occasionally one may observe a few 
beaded forms but these are rare. The more or less regular morphology of the 
pseudo-diphtheria bacillus is quite in contrast to the pleomorphic type of the 
diphtheria bacillus. 

Negri and Mieremet 1 and Bunting and Yates 2 report the successful cultiva- 

1 Centralbl. f. Bakteriol., 1913, LXVIII, 292. 

2 Arch. Int. Med., 1913, XII, 236. See, also, Steele, Boston Med. and Surg. Jour., 1914, 
CLXX, 123; Billings and Rosenow, Jour. Am. Med. Assn., 1913, LXI, 2122; Kusunoki, 
Virchow's Arch., 1914, CCXV, 184; Yates, Bunting and Kristjanson, Jour. Am. Med. Assn., 

1914, LXIII, 2225; Verploegh, Kehrer and van Hoogenhuyze, Munch, med. Wchnschr., 1914, 
LXI, 1158; Mellon, Am. Jour. Med. Sc, 1915, CL, 245; Harris and Wade, Jour. Exper. Med., 

1915, XXI, 493; Fox, Jour. Med. Research, 1915, XXXII, 309 and 325; Arch. Int. Med., 1915, 
XVI, 465; Rhea and Falconer, Ibid., 1915, XV, 438; Bloomfield, Ibid., i9i5,XVI, 197; Bunting, 
Bull. Johns Hopkins Hosp., 1915, XXVI, 179; Olitsky, Jour. Am. Med. Assn., 1915, LXIV, 
1 134; Yates and Bunting, Ibid., 1953; Hatcher and'Lemmon, Ibid., 1915, LXV, 1359. 



CLINICAL BACTERIOLOGY. 711 

tion of a pleomorphic diphtheroid organism in cases of Hodgkin's disease, which 
organism had been previously demonstrated in the tissues by Frankel and 
Much. The former workers used Bordet's potato medium and call the organism 
isolated the Corynebacterium granulomatis maligni, while the latter investigators 
employed Dorset's egg medium and glycerin-phosphate-agar and suggest the 
name of Corynebacterium Hodgkini (Bacillus hodgkini). Rosenow has reported 
the growth of these organisms on Loffler's blood serum and blood agar. The 
morphology of this organism is very pleomorphic: thus plump short rods, some 
so short as to resemble cocco-bacilli; small thin bacilli with polar staining; 
comma-shaped bacilli; granular rods of variable size; branching forms; club- 
shaped involution forms and large spherical types. The organism is Gram- 
positive, is non-acid fast and does not form spores. 

2. Influenza Bacillus (Pfeiffer's Bacillus). 

This organism is found most frequently in the sputum and nasal discharges 
of those affected with influenza. It is often recognizable in the routine sputum 
examinations, but it is often overlooked owing to its small size. 

As hemoglobin is essential for the development of this organism, blood 
agar is the best medium upon which to cultivate it. In obtaining the specimen 
for culture work, select a solid portion of the sputum and wash it several times 
with sterile distilled water to remove the excess of mucus. Or, if desired, make 
an emulsion of the sputum in sterile water. Take up the solid part of the washed 
sputum and rub it over the surface of a blood-agar plate in a Petri dish. Incu- 
bate at 37 C. for 24 or 48 hours. Macroscopic examination shows a number 
of small delicate dew-drop like colonies, which never become confluent. This 
organism is rarely found in pure cultures, the ancillary types being pneumo- 
cocci, streptococci and staphylococci. Other types of colonies will, therefore, 
be seen on the plates. Select a colony having the characteristics above men- 
tioned and prepare smears as usual. The morphology and staining properties 
are as given on page 26. 

3. Pertussis Bacillus (Bordet and Gengou's Bacillus). 

The best medium for the cultivation of this organism is the potato medium 
of Bordet and Gengou, which is prepared as follows. To 200 c.c. of a 4 per 
cent, aqueous solution of glycerin add 100 grams of finely chopped potato. Heat 
in the autoclave for five to ten minutes. The supernatant fluid is now a concen- 
trated glycerin extract of potato. To 50 c.c. of this extract add 150 c.c. of a 
0.6 per cent, sodium chlorid solution and 5 grams of agar. Heat this mixture 
in the autoclave and, while hot, filter it into test-tubes, adding 2 to 3 c.c. to each 
tube. Sterilize in the autoclave. Allow the medium to cool down to 40 to 45 
C. and add an equal amount of sterile defibrinated human or rabbit blood. 
Mix thoroughly and prepare slants of the mixture. This medium is espe- 
cially valuable, also, in cultivating the influenza bacillus, gonococcus and 
meningococcus. 

This bacillus should be looked for in the early stages of tfie disease when 
coughing begins. In the later stages it is not so easily found owing to the large 
number of other bacteria. Select a solid piece of the sputum and wash in 



712 DIAGNOSTIC METHODS. 

sterile distilled water. Spread it over the surface of a Petri plate prepared from 
the above medium. These plates are more satisfactory for the bacilli of per- 
tussis and influenza than are the slants, as these organisms require much 
oxygen. The primary cultures are difficult to obtain, as the growth is so scanty 
during the first 24 hours, and often in 48 hours, that little is visible. Sub-cul- 
tures made from this initial growth give a more copious development. On the 
third day the colonies appear white, slightly elevated and sharply outlined. 
From generation to generation the growth becomes quicker and more luxuriant. 
The morphology and staining characteristics of this organism have been given 
on page 26. 

So close is the similarity of the influenza and the pertussis bacilli that it 
seems advisable to differentiate these organisms. The growth of the former 
is quicker and less luxuriant than that of the latter. The colonies of influenza 
bacilli are slightly bluish and transparent, while those of the pertussis organism 
are whiter and thicker. If stroke cultures are made upon slanted potato-blood- 
agar, the growth of the pertussis bacillus is thicker than that of the influenza 
bacillus; its border is abrupt while that of the influenza bacillus is more spread 
out. The growth of the influenza bacillus is always transparent, shows an 
irregular border and has a moist appearance. Further, the pertussis bacillus 
hemolyzes the blood medium, so that the line of culture appears clearer than the 
surrounding. The influenza bacillus does not cause hemolysis. 

Mallory, Horner and Henderson (Jour. Med. Research, 19 13, XXVII, 391) 
have shown the etiologic relationship of this organism to pertussis, finding it 
in the characteristic lesions between the cilia of the epithelial cells lining the 
trachea and bronchi. 

4. Typhoid Bacillus (Eberth-Gaffky Bacillus). 

The characteristic cultural properties of this organism have been discussed 
on pages 130 and 587, while its agglutination reactions are given on page 607. 
The isolation and detection of the typhoid bacillus in the various excretions and 
secretions of the body is a matter of considerable difficulty, often calling upon all 
the resources at the command of the worker. Study of the cultural character- 
istics, of the agglutination reactions and the staining properties of this organism 
will readily differentiate it from the colon and associated bacteria, but careful 
work is necessary. It is questionable whether the typhoid bacillus has been 
isolated from water and milk supplies in a pure state, but this is not at all neces- 
sary as the detection of the colon bacillus, which is more easily identified, is all 
that is necessary to establish pollution. The typhoid bacillus may be found in 
the dejecta of convalescent and recovered patients for some time, so that care- 
ful measures should be adopted to prevent spread of disease through these 
carriers. 1 

5. Colon Bacillus (Bacillus Coli Communis). 

This organism has been discussed on page 589 and its cultural character- 
istics and points of differentiation given. The colon bacillus is coming into 

1 See Jordan, Jour. Am. Med. Assn., 1914, LXII, 1772; Hirschbrach, Berl. klin. Wchnschr., 
1914, LI, 1176. 



CLINICAL BACTERIOLOGY. 713 

great prominence, owing to its discovery in so many of the secretions and ex- 
cretions of the body under pathological conditions. 1 The use of vaccines 
made from cultures of colon bacilli is varied and the results usually excellent. 

In the bacteriological examination of milk and water, the detection of the 
colon bacillus is of especial importance as an indicator of sewage contamina- 
tion. In times past the custom has been to use, for this work, litmus-lactose 
agar for the plate cultures and glucose broth for the study of the fermentative 
properties of the organisms in the water. It is well known that both water 
and milk, as well as other fluids, contain acid- and gas-producing organisms which 
are not colon bacilli, so that one is not justified in calling all such bacteria 
colon bacilli. In order to eliminate these extraneous gas- and acid-producing 
bacteria, the use of bile has been advocated. The addition of bile to the 
usual media inhibits the other organisms but was not supposed to affect the colon 
bacilli. The media used in this later study and now adopted for all sanitary 
examinations of water and milk supplies are the lactose bile medium, consist- 
ing of 1 per cent, lactose dissolved in ox bile and sterilized, and lactose-bile-agar. 
The bile agar is prepared as follows. In 1,000 c.c. of fresh neutral ox bile dis- 
solve 15 grams of agar, 10 grams of Witte's peptone and 10 grams of lactose, 
boiling as little as possible. When dissolved, filter the medium without titra- 
tion, place in tubes and sterilize in the autoclave for three minutes. Rector 2 
has advocated a slight modification of this medium, which he claims is much 
better. His formula is as follows: 

Dried ox bile, 100 grams 

Witte's peptone, 10 grams 

Agar, 15 grams 

Lactose, 10 grams 

Neutral red (1 per cent, solution), 10 c.c. 

Distilled water, 1000 c.c. 

While it is unquestionably true that this bile medium has an advantage 
over non-bile media in that interfering organisms are eliminated and, there- 
fore, the acid- and gas-producing organisms may more certainly be classified 
as colon bacilli, yet Jordan 3 has shown that this medium exerts an inhibiting 
effect on colon bacilli themselves to the extent of 33 1/3 to 50 per cent, of the 
viable colon bacilli present in the material under investigation. Such being 
the case much more care must be exercised in reporting upon a specimen of 
water or milk, especially as to numbers of colon bacilli and as to the safety of 
such specimens for general use. 

6. Dysentery Bacillus (Shiga-Kruse Bacillus). 

In bacillary dysentery certain organisms are found which closely resemble 
the typhoid and colon bacilli in morphology but which differ in certain cultural 

1 See Conradi and Bierast, Kolle and Wassermann's Handbuch, 1913, VI, 483; also 
Bornand, Centralbl. f. Bakteriol., Abt. 2, 1913, XXXVIII, 516; Kligler, Jour. Infect. Dis., 
1914, XV, 205; Browne, Ibid., 580; Rogers, Clark and Davis, Ibid., 1914, XVI, 411; Clark and 
Lubs, Ibid., 1915, XVII, 160. 

2 Am. Jour. Pub. Health, 19 13, III, 154. 
8 Jour. Infect. Dis., 1913, XII, 326. 



714 



DIAGNOSTIC METHODS. 



characteristics from the latter bacteria. In the course of an epidemic of 
dysentery in Japan, Shiga discovered this bacillus, but it remained for Kruse 
to elaborate and fix its characteristics. Since that time, similar organisms have 
been found in various epidemics throughout the world, but in many cases certain 
differences were observed in the behavior of the organisms toward different 
sugar-containing media. On the basis of fermentative tests with acid produc- 
tion in sugar-broths, and, also, upon variability in the agglutination reactions 
of the organisms isolated, at least four distinct types of the dysentery bacillus 
must be considered as established. 



I. Shiga-Kruse type. 
II. Flexner-Strong type. 

III. Hiss-Russell, El Tor I strain, 

IV. Harris- Wollstein type. 



Y" type. 



The following table, taken from Jordan, 1 will show at a glance the variations 
in the action of these similar organisms. 



Bacillus dysenterise 




Acid production from: 




Indol 


Mannite 


Maltose 


Saccharose 


Dextrose 


production 


Type I (Shiga-Kruse) 

Type II (Flexner-Strong) .... 

Type III (Hiss-RusseU) 

Type IV (Harris- Wollstein) . . . 


+ 
+ 
+ 


111 + 


+ 
+ 


+ 
+ 
+ 
+ 


+ 
+ 

+ 



The detection of these organisms in the stool is not a matter of great diffi- 
culty, by cultural methods, if the case is examined relatively early in its progress. 
However, after some time the examination becomes more and more uncertain 
until the organisms are rarely found after the third week. The material for 
the preparation of cultures is obtained from a freshly passed stool. Flecks 
of mucoid material are selected and placed in sterile water or physiologic salt 
solution, in order to remove the colon bacilli with which the material is con- 
taminated. This washing is repeated three or four times, fresh water being, of 
course, added each time. If there be no visible mucus in the stool, emulsify 
a portion of the stool in nutrient broth. The washed mucoid particles, or a 
portion of the fecal emulsion, are now rubbed over the surface of three or four 
litmus-lactose agar plates or of plates made of Drigalski and Conradi's medium 
(see p. 130) without the addition of the crystal violet. Allow these plates to 
dry in the air by keeping the cover slightly open for one-half hour, place them 
in the incubator at 37 C. with the bottom side up and examine at the end of 
24 hours. Any colon bacilli left after the washing, and there are always some, 
will appear as red colonies with a gradual shading of color into the blue medium. 

1 General Bacteriology, Philadelphia, 1913. See, also, Lentz, Kolle and Wassermann's 
Handb., 1913, III, 927; Barber, Phillipine Jour. Sc, 1913, VIII (B), 539; Bluhdorn, Deutsch. 
med. Wchnschr., 1914, XL, 782; Monatsschr. f. Kinderhkde., 1914, XIII, Orig., 37; Schild, 
Ibid., 51; Musgrave and Sison, Philippine Jour. Sc, 1914, IX (B), 241; Flexner and Amoss, 
Jour. Exper. Med., 191 5, XXI, 515. 



CLINICAL BACTERIOLOGY. 715 

The Shiga bacillus does not change the color of the medium, its colonies ap- 
pearing as translucent, iridescent, dew-drop like growths with irregular margins 
and centers more opaque than the edges. This growth is very similar to that of 
the typhoid bacillus on this medium. Test one of the latter colonies for motility 
of the organisms by the hanging drop method and, also, test its agglutination 
with a potent dysentery serum. If these tests are satisfactory, namely, if no 
motility is observed and agglutination is definite, sub-cultures of these colonies 
are made upon slant agar, neutral red-glucose- agar (glucose agar to which three 
or four drops of a saturated aqueous solution of neutral red are added), litmus- 
milk and plain milk. The cultures on agar and neutral red- glucose- agar 
show the same characteristics as do those of the typhoid bacillus, but the lack 
of motility excludes this latter organism. These cultures have a peculiar sperm- 
like odor. On litmus-mannite-agar the Shiga bacillus produces no acid, while 
the typhoid bacillus develops a distinct red color. Litmus-milk is not changed 
in color, nor is plain milk coagulated. 

It will be seen, therefore, that the Shiga bacillus differs from the typhoid 
bacillus in its action toward litmus-mannite-agar and, especially, in its lack of 
motility and its different agglutination reactions. From the colon bacillus, 
the cultural characteristics differentiate it sharply. 

The morphology and staining properties of this organism have been given 
on page 132. It is Gram-negative, does not form spores, nor does it possess 
flagella. In smear preparations, the dysentery bacillus resembles the typhoid 
bacillus, but the former is thick and often polymorphous. 

Culturally the other types of dysentery bacilli are differentiated from the 
Shiga bacillus by the fact that their cultures on litmus-lactose- agar are more 
irregular and have a reddish-violet tinge, while the latter organism shows colonies 
of a translucent dew-drop like appearance. Further, the Shiga bacillus does 
not produce indol, while all the other types, as well as the colon bacillus, do. 
In confirming this differential diagnosis, the agglutination tests are valuable. 
The Shiga bacillus should agglutinate in a dilution of 1 to 20 of the patient's 
serum, while the Flexner type agglutinates at a dilution of 1 to 80. For dif- 
ferentiation of the other types, the fermentative tests must be utilized, as 
agglutination is not typical. 

7. Cholera Vibrio (Koch's Comma Bacillus). 

This organism has been briefly discussed on page 129. It appears in stained 
preparations as a small, short slightly curved rod, which has led to the name of 
" comma" bacillus. It is about 2 ^ long and 0.5 /x broad. It is not infrequent 
to observe long, straight involution forms, especially from liquefied gelatin cul- 
tures. This bacillus has a single flagellum at one end and shows very active 
motility. It stains easily with the ordinary stains and is Gram-negative. 

The spirillum of cholera grows well on practically all media at room tem- 
perature, with the exception of potato. The medium must be alkaline in re- 
action to insure the growth. This alkalinity is obtained by adding 3 c.c. of 
10 per cent, sodium hydrate to 100 c.c. of neutral medium. 

The growth on gelatin plates at room temperature is more or less character- 



716 DIAGNOSTIC METHODS. 

istic. These colonies appear, after 24 hours, as round and regular white points, 
which later become irregular and show a granular appearance as if the colony 
were covered with ground glass. Within 48 hours liquefaction begins, the 
colony sinking into the depression. On gelatin stab-cultures, the liquefaction 
begins as a cup-shaped depression at the surface, a bubble of air being retained 
in the depression. The later liquef action takes the form of a funnel, being much 
broader at the surface. Later the medium is entirely liquefied. Some of the 
vibrios resembling the cholera bacillus, as especially the Finkler-Prior vibrio, 
produce a more rapid liquefaction of the gelatin and do not show the bubble of 
air which is practically always present in the cholera cultures. This point is not, 
however, distinctive enough for strict diagnostic purposes. It is to be remem- 
bered, in this connection, that the other types of organisms found in the intes- 
tinal canal do not liquefy gelatin media. 

On agar plates the colonies appear as pale, flat, thin growths which are trans- 
parent and show, by transmitted light, a peculiar opalescence. As the cholera 
spirillum is a strongly aerobic organism, we find a copious surface growth in 1 per 
cent, peptone solution containing 1/2 per cent, sodium chlorid. Other organ- 
isms do not develop readily in this medium, so that we not infrequently may 
obtain an almost pure culture by use of this simple medium. The addition of 
a few drops of concentrated sulphuric acid to such a culture produces a bur- 
gundy-red color, due to the formation of nitroso-indol. Thus the cholera 
spirillum produces indol and, at the same time, reduces the nitrates of the culture 
medium to nitrites. While the colon bacillus produces indol, it is necessary 
to add a trace of nitrite before the nitroso-indol reaction is obtained. This 
cholera-red reaction, as it is called, was supposed to be distinctive for the cholera 
vibrio but it has been shown that other cholera-like organisms also react. A 
negative result with this test is, therefore, of greater value than a positive one. 

In the bacteriological diagnosis of cholera, several points must be borne in 
mind. Select some of the "rice particles" and inoculate a peptone water cul- 
ture as above, or plant 1 c.c. of fecal material in 50 c.c. of this medium. From this 
surface growth, at the end of six to eight hours at 37 C, sub-cultures are made 
upon other media and smears are examined and studied for the characteristic 
organisms. Glycerin and agar plates are inoculated and incubated for 24 to 
48 hours, the former at room temperature and the latter in the incubator. It 
should be a rule to inoculate Dieudonnes alkali-blood-agar at the same time. 
This medium is prepared by mixing equal parts of defibrinated beef blood and 
normal (4 per cent.) sodium hydrate ; heat in autoclave for one-half to three-fourths 
hour. Add 30 parts of this mixture, while still hot, to 70 parts of neutral 3 per 
cent. agar. Mix and sterilize in the autoclave. When cooled, pour into Petri 
plates. Allow the plates to dry by standing open in incubator for several hours 
and do not use these plates earlier than 24 hours after preparing. Colon bacilli 
and other extraneous organisms do not grow on this medium, while the cholera 
organism develops rapidly. The colonies are large, circular, crystal-clear by 
transmitted light and by reflected light gray. Microscopic examination of the 
organisms from these colonies show many degenerative forms, very rarely any 
typical comma forms. 



CLINICAL BACTERIOLOGY. 717 

After these cultures are prepared test the motility of the organism in hanging 
drop and follow this by the agglutinating reactions and, especially, by the 
application of Pfeiffer's bacteriolytic test. This latter test is as follows: An 
emulsion of the organisms in question is injected into the peritoneal cavity 
of a guinea-pig, which has been immunized against a definite species of the 
vibrio. In 20 to 30 minutes remove, with a trocar, some of the peritoneal fluid 
and examine under the microscope. The organisms present will be non- 
motile and appear as more or less granular spherical masses instead of the 
typical forms, if the reaction is positive; if negative, the organisms will appear 
as in the usual preparations. 1 

8. Gram-positive Cocci. 
A . Pneumococcus (Diplococcus of Fraenkel ; Streptococcus Pneumoniae) . 

This organism is practically always found in the normal throat, so that its 
presence therein does not indicate a pathologic process. It is found, also, as 
the primary etiologic factor, in a wide range of infectious processes, so that it 
must not be regarded as limited to pneumonic types of infection. The mor- 
phology and staining characteristics of this organism have been given on page 
25, while its cultural peculiarities, especially, have been discussed on page 589. 
It is to be remembered that, from the morphological standpoint, it is oftentimes 
impossible to distinguish the pneumococcus from the streptococcus, as the former 
not infrequently appears in chains, and the typical lanceolate diplococci give 
place to forms which are more nearly true coccoid types. 

Cultural methods must, therefore, be employed and the special pe- 
culiarities of the pneumococcus and streptococcus differentiated by the use 
of the blood- agar plates, the blood bouillon tubes, the use of bile acids, and 
the fermentative test with inulin as outlined on page 590. The agglutina- 
tion test should, also, be employed with specific immune serum in all cases 
of doubtful diagnosis. 2 

It is to be remembered that there are distinct varieties of pneumococci, 
which are distinguishable from one another only by careful study of the cul- 
tural peculiarities as well as by observation of their effects on animals. Transi- 
tion forms from the pneumococcus to the streptococcus and vice versa are ob- 
served under certain cultural conditions, so that it is often a more than difficult 
problem properly to classify these organisms. One of these organisms, the 
streptococcus mucosus capsulatus, is probably to be considered with the true 
pneumococci. It occurs as round, or very slightly lance-shaped cocci, which 
are encapsulated, the capsule being wider than that of the pneumococcus. The 
organisms usually appear in pairs, but there are always seen a few in chains of 
four to eight organisms. The colonies on blood agar have a mucoid slimy 
consistency and have a greater tendency to become confluent than do those of 
the true pneumococcus. 

1 See "The Bacteriological Diagnosis of Cholera," Pub. Health Rep., 1912, XXVII, 371; 
also, Kolle and Schiirmann, Kolle and Wassermann's Handb., 1912, IV, 1; Kabeshima, 
Centralbl. f. Bakteriol., Abt. 1, 1913, LXX, 202;Craster, Jour. Exper. Med., 1914, XIX, 581. 

2 See Dochez and Gillespie, Jour. Am. Med. Assn., 1913, LXI, 727; also, Floyd and Wol- 
bach, Jour. Med. Research, 1913, XXIX, 493; Dochez and Avery, Jour. Exper. Med., 191 5, 
XXI, 114; Lyall, Ibid., 146. 



718 DIAGNOSTIC METHODS. 

B. Streptococci. 

There are several varieties of streptococci, which are not readily distin- 
guished by their morphology but which cultural peculiarities usually suffice to 
identify. These cultural points have been discussed on pages 590 and 591. 
It is to be said, however, that this differentiation is not a simple proposition, 
as these varieties readily change, the one into the other and, also, into forms 
which are hardly distinguishable from the typical pneumococcus. The chief 
types of the streptococcus are 

a. Streptococcus pyogenes {hemolyticus) , along-chained form which produces 
no green coloration on blood agar but shows a large colorless hemolytic area 
about its colonies. 

b. Streptococcus viridans, a short-chain type producing green colonies on 
blood agar but only slight hemolysis. 

c. Streptococcus mucosus, an encapsulated organism discussed above, pro- 
ducing no green colonies nor any hemolysis. 

Many other types have been isolated in pathological conditions and their 
exact classification is rather difficult. Usually the organisms have shown char- 
acteristics which place them in classes intermediate between those given above 
and even have indicated their close relationship to the pneumococcus. One 
of these virulent organisms has been discussed on page 40, being called the 
streptococcus epidemicus and causing streptococcic sore-throat. 

In these later days much work has been done on this group of organisms 
and much of value has been forthcoming. Some of this work is so important 
that the writer must discuss it briefly, in order properly to correlate the subject 
of streptococci to various pathological processes. 

Davis 1 has called attention to the close association of streptococcic infec- 
tions to chronic arthritic troubles and finds that the atrium of infection in these 
arthritides is usually the tonsil or peritonsillar structures. As is demonstrated 
the streptococcus hemolyticus, the diplococcus (streptococcus) rheumaticus 
and the streptococcus epidemicus (see p. 40) are very prone to attack the joints; 
while the streptococcus viridans rarely does so. Such cases can be separated by 
the clinical findings and history of tonsillitis in the patients from the other types 
of deforming arthritides. The bacteriologic examination of cultures from the 
tonsil clears up the etiology. In taking the cultures from the tonsil, the 
material must be obtained from the depths of the crypts after the tonsil has 
been incised. In such cultures the streptococcus hemolyticus is usually found. 
The streptococcus viridans is found in surface cultures, but this is disregarded 
as it is less virulent and is rarely associated with arthritic involvements. It is 
to be recalled that the streptococcus hemolyticus may be found on the surface 
of the tonsils in ordinary streptococcic tonsillitis, and even on normal tonsils, 
but these do not necessarily have any special general value in the absence of 
clinical history of joint troubles. In the treatment of these cases of arthritis 
due to streptococcic infection, proper vaccines often work like magic. Two 

1 Jour. Infect. Dis., 1912, X, 148; Jour. Am. Med. Assn., 1913, LXI, 724. See, also, 
Richards, Ibid., 1914, LXII, no; Holman, Jour. Path, and Bacterid., 1915, XIX, 478. 



CLINICAL BACTERIOLOGY. 719 

hundred million of the killed organisms are given at a dose with five-day intervals 
between the administrations. 

Rosenow 1 in his work on the pneumococcus and streptococcus has isolated 
certain organisms from infected joints, blood cultures and tonsillar infections 
in cases of acute articular rheumatism and finds organisms corresponding to the 
micrococcus rheumaticus of Poynton and Payne. He finds, however, that 
this organism resembles very closely the streptococci, its virulence being less 
than that of the streptococcus hemolyticus and greater than that of the strepto- 
coccus viridans. By animal passage and other means three of the strains iso- 
lated from acute rheumatism have been converted into typical hemolytic strepto- 
cocci on the one hand and into pneumococci on the other. The cultures made 
from the involved joints and regional lymph nodes have revealed three types of 
streptococci. One produces green colonies on blood agar and forms long chains; 
a second produces a narrow zone of hemolysis and forms short chains; while the 
third forms a grayish-brown colony without affecting perceptibly the blood 
in the medium. This latter organism appears as a diplococcus, in short chains 
and as single cocci. The most distinctive cultural feature of these types is their 
production of a very high acid reaction in dextrose-broth and abundant growth 
at low temperatures. Progressive transmutation from a streptococcic type to 
a pneumococcus is easily shown. 

Billings, 2 on the basis of this work of Davis and Rosenow, has studied the 
clinical aspects of arthritic cases and finds that a chronic focal infection is as- 
sociated with many of these arthritides. Clinically, he finds the focal disease 
in the faucial tonsil, dental alveoli or jaw, antra of head, etc., in a large number 
of cases. In other cases chronic colon, gonorrheal or streptococcic infection of 
the seminal vesicles and prostate were found to be the focal point of the arthritic 
involvement. Cultures were prepared from the material obtained from the 
focal infection, sub-cultures made and the dominant organism isolated in pure 
culture. From these cultures vaccines were prepared and used with excellent 
results. Billings is especially emphatic upon the point of using the auto-vaccine 
from the dominant isolated organism and believes that only by this method may 
good result from vaccine treatment. 

C. Staphylococcus Pyogenes. 

These organisms grow well on all types of media. Their cultures on agar 
are best adapted to the development of the pigment, which is the characteristic 
differentiating point of members of this species. Cultivation at room tempera- 
ture frequently brings out the pigment better than growth in the incubator. 
These organisms are readily recognized, in stained preparations, by their mor- 
phology. They appear as distinct cocci, five to fifty in a cluster often com- 

1 Jour. Am. Med. Assn., 1913, LX, 1233; Jour. Infect. Dis., 1914, XIV, 1 and 61; Rosenow, 
New York Med. Jour., 1914, XCIX, 270; Jour. Am. Med. Assn.. 1914, LXII, 1146; Ibid., 1914, 
LXIII, 903; Davis, 111. Med. Jour., 1914, XXVI, 158; Billings, Ibid., 164; Jour. Am. Med. Assn., 
1914, LXIII, 899; Smith and Brown, Jour. Med. Research, 1914, XXXI, 455; Hopkins and 
Lang, Jour. Infect. Dis., 1914, XV, 63; Thro, Ibid., 234; Holman, Ibid., 293; Crabtree, Ibid., 
309; Davis, Ibid., 378; Rosenow, Ibid., 1915, XVI, 367; Thro, Ibid., 1915, XVII, 227; Broad- 
hurst, Ibid., 277; Brawley, 111. Med. Jour., 1915, XXVIII, 178. 

2 Arch. Int. Med., 1912, LX, 484; Jour. Am. Med. Assn., 1913, LXI, 819. 



720 DIAGNOSTIC METHODS. 

pared to a bunch of grapes. Rarely do they occur singly or in pairs. Oc- 
casionally short chains may be observed, but most of them are clumped. In 
purulent material these organisms may be both intra- and extracellular, but 
their staining characteristics readily distinguish them from the Gram-negative 
types of organism. 

9. Gram-negative Cocci. 
A. Gonococcus (Diplococcus of Neisser). 

The morphological and staining characteristics of the gonococcus have been 
discussed on page 668. It is to be emphasized that this organism cannot, in 
every case, be distinguished by its morphology, staining characteristics and 
intracellular position from other organisms, especially from the meningococcus, 
micrococcus catarrhalis and a few saprophytic diplococci, such as diplococci 
rlavus, crassus and cinereus. 1 In judging of the possibility of a certain organism 
being the gonococcus, the origin of the culture must be known and something 
of the clinical history must be given. Further, it is to be recalled that the 
intracellular grouping of the organisms is not especially limited to the gonococcus 
nor is it always present in every gonorrheal case. Such arrangement is given 
both by the meningococcus and micrococcus catarrhalis. In the early cases of 
gonorrhea, when pus cells are very few in number, the gonococci are for the most 
part extracellular, some of them lying upon the epithelial cells, but practically 
none being found within the pus cells. As the pus cells increase in number, 
the intracellular types become predominant owing to the active phagocytosis. 
In the later stages and in the chronic forms of infection, the extracellular types 
become more frequent, very few intracellular forms being observed. 

As the gonococcus does not grow readily, if at all, on the ordinary culture 
media, special media is necessary. Albumin, preferably of human origin, is 
essential in any medium to be used. The best medium is Wertheim's serum 
agar prepared by adding 1 part of human serum to 2 to 3 parts of nutrient agar. 
The serum is warmed to 40 C. and added to the liquefied agar of the same 
temperature. Instead of the serum, ascitic or hydrocele fluid may be substituted 
as suggested by Kiefer and Steinschneider. In fact, ascitic agar is probably 
more frequently used than is the serum agar. Blood agar also affords an ex- 
cellent medium for the development of the gonococcus. 

On the above media, colonies of the gonococcus appear in 24 hours as gray- 
ish-white points, which merge later into growths of a grayish-blue tinge with 
slight iridescence. These colonies have a slimy consistency, are round but soon 
become confluent. The gonococcus is very sensitive to drying, so that the cul- 
ture medium must have plenty of water of condensation. It survives exposure 
to air for only a short time, although in masses of dried pus it may live, excep- 
tionally, for six or seven weeks. In favorable culture media it rarely maintains 
its vitality more than 48 to 72 hours at room temperature but will live longer if 
kept in a refrigerator (Jordan). 

The gonococcus is bacteriologically identified by its characteristic morphol- 
ogy, its Gram-negative staining, its frequent intracellular position and by its 
ready growth on blood or ascitic agar and its lack of growth on ordinary media. 
1 See Kligler, Jour. Exper. Med., 1913, XVII, 653. 



CLINICAL BACTERIOLOGY. 72 1 

These characteristics are, however, not sufficient to differentiate it from the 
closely related organisms, which will be discussed below. It is to be said that a 
diagnosis of a gonorrheal infection outside of the genital tract is never safe if 
based entirely on microscopic evidence, and the cultural evidence is not always 
clear. The micrococcus catarrhalis is not infrequently the etiologic factor in a 
genito-urinary infection as well as in other parts of the system, while the meningo- 
coccus very often causes certain genito-urinary complications, which may be 
associated with the presence of this latter organism in this tract (see Koch 1 ). 
Further, in chronic cases of true gonorrhea, it is very frequently a difficult matter 
to detect the gonococcus either microscopically or culturally. The organisms 
found may be Gram-variable and few will be intracellular, so that great care 
must be exercised in diagnosis. In such cases one should use the provocative 
test of administering some irritant or allowing the patient to partake freely of 
alcohol. Such a procedure followed by milking the prostate will usually 
result in the appearance of typical gonococci in the purulent material expressed. 
Of course in these cases one may use the complement-fixation test or the vaccina- 
tion test as suggested by Irons and, later, by Fronstein (see p. 604). 

From the clinical point it may tax the resources of the physician to establish 
a diagnosis of gonorrhea in any given case, outside of those cases of acute 
gonorrhea in which the history and symptomatology are clear. However, in 
cases of meningitis with genito-urinary complications or of general systemic 
gonorrheal infection with meningeal symptoms, the diagnosis is not so simple, as 
a differentiation must be established between the meningococcus and 
gonococcus. 

In this differentiation, cultivate the material obtained from the urethral dis- 
charge or from lumbar puncture upon faintly alkaline ascitic agar for 24 to 48 
hours. The meningococcus grows more luxuriantly than the gonococcus. The 
colonies of the former are larger (2 to 3 mm. in diameter) than those of the latter 
(1 to 1.5 mm.). On microscopic examination of these colonies with the low 
power, the colonies of the meningococcus are found to be flat, homogeneous, 
usually round, very slightly irregular border but never notched; while those of 
the gonococcus are irregular, border usually notched and center always elevated. 
On cultivating these organisms on litmus-ascitic-agar media containing various 
sugars, the gonococcus will show acid production in the dextrose media only; 
while the meningococcus produces acid in both dextrose and maltose, but in no 
others. 

The micrococcus catarrhalis, with which the gonococcus may be often con- 
fused, grows well on ordinary media. It does not produce acid in any sugar- 
containing medium. Its morphology, staining characteristics and intracellular 
position are, however, exactly like those of the gonococcus. 

B. Meningococcus (Diplococcus Intracellularis Meningitidis) . 

The morphological and staining characteristics of this organism of Weichsel- 
baum have been given on page 682. For the bacteriological diagnosis of a 
meningococcic infection, one may use spinal fluid, blood, or purulent products 

1 Kolle and Wassermann's Handbuch, 1913, IV, 68g. 
46 






72 2 DIAGNOSTIC METHODS. 

from metastatic points, secretions of nose and bronchi. This material should be 
examined as soon as possible after obtaining, as the meningococci are not espe- 
cially resistant to various changes. 

For the development of this organism, especially of the first generation, 
media are necessary which contain animal, preferably human, albumin. The 
blood agar or ascitic agar given above are the best to use. The cultural pecul- 
iarities of the meningococcus on this medium have been given. From the cul- 
tures the organisms not infrequently appear variable in size and show variable 
depth of staining, although they are always Gram-negative. 

In examining spinal fluid for these organisms, centrifuge the fluid in a sterile 
tube and plant two or three ascitic agar plates with the sediment. Isolate the 
colonies by sub-culture if necessary and identify the organism microscopically. 
Inoculate special sugar-containing media and differentiate by the results men- 
tioned above. The meningococcus produces acid only in dextrose and maltose 
media. 

In examining material from the nose or throat simple microscopical ex- 
amination is of little avail, as we have the possibility of finding the micrococcus 
catarrhalis as well as the saprophytic diplococci previously mentioned. Cul- 
tural work and differentiation on sugar media are absolutely essential. These 
saprophytic cocci, especially the diplococcus crassus, are found in both the 
lumbar fluid and nasal secretions. This latter diplococcus produces acid in 
dextrose, maltose, galactose, levulose, cane-sugar and lactose media. This or- 
ganism is probably identical with Jagers meningococcus. Some of the varieties 
of the diplococcus flavus ferment levulose in addition to dextrose and maltose, 
while other types (Diplococcus flavus III) acts exactly like the meningococcus. 1 
It will be seen, therefore, that the fermentation test should be applied in all 
differential work, where the meningococcus is concerned. 

C. Micrococcus Catarrhalis. 

This organism is discussed on page 17. It is found in catarrhal conditions 
of nose and throat and, also, in infections of the genito-urinary tract. It grows 
readily on all media. On ascitic agar plates the colonies are white, firm, irregu- 
lar, dry and somewhat smaller than the growths of meningococcus. Under low 
microscopic power they appear brown and granular, while those of the meningo- 
coccus are yellow and homogeneous and of the gonococcus grayish and slimy. 
The firm and dry quality of the colony is characteristic of the micrococcus catar- 
rhalis. From the ordinary staphylococci, it is differentiated by its non-lique- 
faction of gelatin. 

Preparations made from culture media show involution forms, some of the 
organisms being very large. In the direct smears of purulent material the organ- 
isms are Gram-negative, intracellular or extracellular biscuit-shaped diplococci. 
For its identification cultural work is necessary and a study of its fermentative 
powers is essential. The micrococcus catarrhalis does not produce acid in any 
sugar-containing medium. An organism described as the Micrococcus cinereus 
is probably identical with this organism of catarrh. 

1 See Kutscner, Kolle and Wassermann's Handbuch, 1913, IV, 5S9. 



CLINICAL BACTERIOLOGY. 723 

VIII. Vaccines. 

In previous sections of this book the problems of immunity in its relation to 
opsonins, agglutinins and precipitins have been discussed briefly. It was shown 
that the injection of a vaccine (a suspension, in physiologic salt solution, of a 
certain number of killed organisms) into the body of a patient suffering with a 
chronic infection due to the organism injected brought about a systemic reaction 
by which the resistance of the patient was increased. In this way it was hoped 
that the natural resources of the body would be brought into activity by the 
stimulation of antibody formation and that this would result in great benefit to 
the patient. Further, by the use of vaccines, the resistance of the human organ- 
ism against bacterial invasion could be so increased that immunity was conferred, 
for a variable period of time, toward the organism so injected. The most recent 
use of this latter principle is the antityphoid vaccination, which will be discussed 
later. 

It will be remembered that patients, who have passed through an attack of 
an infectious disease, have acquired, in most infectious diseases, an immunity 
against reinfection. An attempt is often made, especially in the treatment of 
infections with diphtheria organisms, tetanus bacilli and meningococci, to aid 
the patient in his efforts to overcome the invasion, that is to help him in acquiring 
immunity, by injection of serum from an animal previously immunized against 
such infection. This serum or antitoxin supplies antibodies to the infected 
patient, with which he may overcome the toxic products of bacterial develop- 
ment in the system. In other words, serum therapy confers immunity, shown 
in recovery from the acute infection, by aiding the system through introduction 
of antibodies produced outside the system of the patient. Vaccine therapy, on 
the other hand, produces immunity by causing the production of antibodies 
within the system itself. It must be recalled that patients who have been in- 
oculated with serum (protein) often become sensitized to its action to such an 
extent that a later injection of the same serum, even in much smaller dosage, 
may provoke serious and even fatal results. This is the phenomenon of anaphy- 
laxis and must be remembered whenever one is dealing with the parenteral 
introduction of protein material into the system. 1 

In this section the writer wishes to discuss the method of preparation of 
vaccines, both of the stock and autogenous varieties. The elaboration of the 
principles upon which their use is based as well as a discussion of the thera- 
peutic value finds no place here. 

1 See Besredka, in Kraus and Levaditi's Handbuch, 1911, Erganzungsbd. I, 209; Biedl 
and Kraus, Ibid., 255; Abderhalden, Ztschr. f. physiol. Chem., 1912, LXXXII, 109; Wells and 
Osborne, Jour. Infect. Dis., 19 13, XII, 341; Weil, Jour. Med. Research, 19 13, XXVIII, 243; 
Auer and Van Slyke, Jour. Exper. Med., 1913, XVIII, 210; Zinsser, Ibid., 219; Vaughan, Jour. 
Am. Med. Assn., 1913, LXI, 1761; Friedberger, Nederl. Tijdschr. v. Geneesk., 1913, II, 
1065 an d 1278; Nixon, Brit. Med. Jour., 1913, II, 1351; Weil, Jour. Med. Research, 1913, 
XXLX, 233; Leschke, Ztschr. f. exper. Path. u. Therap., 1914, XV, 23, Seligmann, "Anaphy- 
laxie," in Handb. d. Biochem. des Menschen u. d. Tiere, Erganzbd., 1913, 24S; Vaughan, 
"Anaphylaxis and Infection," Harvey Lectures, 1913-14, p. 132; Bradley and Sansum, Jour. 
Biol. Chem., 1914, XVIII, 497; Wallace, Jour. Am. Med. Assn., 1914, LXII, 1166. 



724 DIAGNOSTIC METHODS. 

i. Preparation of Vaccines. 
A. Stock Vaccines. 

The material for this work is obtained, under the strictest precautions to 
prevent contamination, from a focus of infection with the organism whose vaccine 
is desired. It is at once plated upon general or special medium, depending upon 
the organism in question, and the organism isolated in pure culture. This may 
be a simple matter or it may be a very complicated one. In any case, all the 
differential points previously given for the varying organisms must be kept in 
mind, so that proper medium and methods of isolation may be employed. It is 
absolutely essential that the final culture be pure, if the vaccine is to be of any 
value. 

In order to obtain a large amount of the pure culture, transfers from the 
original pure culture are made to three or four tubes of the medium best suited 
for its development and these tubes incubated for 24 to 48 hours. The cultures 
are then washed from the surface of the medium, using sterile physiologic salt 
solution as the menstruum and scraping the surface lightly with the platinum 
needle to insure the removal of the organisms. Pour this suspension into a 
sterile flask or large tube and shake thoroughly to break up the bacterial masses. 
This is an important point in the technic, as otherwise the suspension will not 
be uniform. If necessary, one may filter out the large clumps by means of a 
sterile filtering arrangement. 

Having prepared the suspension, this is then sterilized by heating in a water- 
bath to 6o° C. for one hour. From this tentatively sterilized suspension, cultures 
are made upon proper media by transferring several loopsful of the suspension 
to the surface of the slanted media and incubating for 48 hours. If no growth 
occurs, the suspension may be regarded as sterile. 1 

Standardization. — The sterilized vaccine is now examined for the number of 
bacteria per c.c. of the suspension. The method usually adopted is to compare 
the number of bacteria in a definite volume of suspension with the number of 
red blood cells in the same volume of freshly drawn blood. The technic is as 
follows. By means of a graduated capillary pipet place equal volumes of 
blood and of bacterial suspension on a glass slide, mix thoroughly and spread as 
for an ordinary blood slide. Stain with Wright's or other blood stain. Count 
the number of red cells and of bacteria in a number of microscopic fields and 
obtain an average of each. As the number of red cells per cu. mm. is arbi- 
trarily fixed at 5 million, the relation of bacteria to the red cells, as shown by 
the counts, will readily give the number of bacteria in the cu. mm. The number 
in 1 c.c. is obviously 1,000 times greater. 2 

Having obtained the concentration of the original suspension, the vaccine 
must be standardized so that each c.c. shall contain a standard number of 

1 King and Davis (Am. Jour. Pub. Health, 1914, IV, 917) have shown that potassium 
tellurite is an excellent indicator of sterility. Characteristic black compounds are produced 
if a little of the salt be added to fluids containing viable bacteria. 

2 See Hopkins (Jour. Am. Med. Assn., 1913, LX, 1615) for a method of standardizing 
vaccines based on centrifugation and determination of percentage of bacterial sediment; Glynn, 
Powell, Rees and Cox, Jour. Path, and Bacterid., 1914, XVIII, 379; Blaker, Indian Jour. Med. 
Research, 1914, I, 726; Harvey and Acton, Ibid., 1914, II, 648; Fitch, Jour. Am. Med. Assn., 
1915, LXTV, 893. 



CLINICAL BACTERIOLOGY. 725 

bacteria. This is done by diluting with sterile physiologic salt solution (which 
contains sufficient cresol to bring the concentration of the final product up to 
0.2 to 0.4 per cent, cresol) until the proper strength is reached. The addition 
of the cresol prevents future contamination of the vaccine and is an additional 
safeguard as to sterility of the product. The standard maximum dosage per c.c. 
of some of the more usual stock vaccines is as follows: 

Staphylococcus, 500 million 

Streptococcus, 100 million 

Colon bacillus, 100 million 

Gonococcus, 100 million 

Typhoid, 1,000 million 

Pneumococcus, 100 million 

Of course this maximum is not suited to all cases. The dosage must be varied 
for the individual and for the type of infection. 

B. Mixed Stock Vaccines. 

A prerequisite to the use of any vaccine should be the bacteriological identi- 
fication of the infection in question. The use of a simple or mixed stock vaccine 
is never justified without such a precaution. It is questionable whether one 
should ever use a mixed stock vaccine, but if the bacteriological examination has 
revealed a mixed infection, a proper mixture may be used in the proportions 
suitable for each separate constituent of the vaccine. In recent years the 
number and varieties of mixed stock vaccines have increased to such an extent 
that they are to be regarded as questionable therapeutic agents. To quote from 
Billings, "There is a value in vaccine therapy but to obtain benefit from vac- 
cines, they should be rationally used. To use vaccines, simple or mixed, in the 
treatment of a patient, without first ascertaining the nature of the disease, and, 
if infectious, the kind of invading organism, is unscientific, reprehensible and 
wrong. When the mixed vaccines or the filtrate of culture-broth of countless 
organisms are used, it is like the shotgun prescription of our ancestors of the 
profession. They were and are intended to hit something." Such usage must 
be regarded, therefore, as mere guesswork. 

C. Autogenous Vaccines. 

These are the proper agents to use whenever a vaccine is to be employed. 
The specific toxins of the organism call into activity the specific antibodies, which 
are necessary to overcome the effects of the organism causing the infection, for 
which the autogenous vaccine is being prepared. " Instead of adding a heavier 
burden to the individual's immunizing mechanism, already taxed in its struggle 
against the invading bacteria, these bacteria, reintroduced as autogenous vac- 
cines, if successful stimulate further therapeutic immunizing responses." It 
can be considered as established that these autogenous vaccines are the ones 
giving the best results. 

These autovaccines are prepared from the discahrges, excretions, blood, etc., 
of the patient. The effort to obtain pure cultures and definitely to identify the 
organism or organisms which are dominant must be painstaking, both aerobic 



726 DIAGNOSTIC METHODS. 

and anaerobic cultures being made in many cases. If there be a mixed infection, 
each organism is isolated in pure culture and the suspensions of the pure cultures 
are then mixed in such a way that the resulting combination will contain the 
organisms in their usual strength per c.c. In this standardization of the auto- 
genous vaccines a little more latitude is allowed than in the case of stock 
vaccines, as the dosage of the autovaccine must be adjusted more to the in- 
dividual than to the standard. In other words, one must use these autovaccines 
in graduated dosage, beginning with a small dosage and working up. 

It is true that occasionally mixed autovaccines are prepared which have not 
been sufficiently differentiated. This method is uncertain and crude. The 
method employed is to obtain a culture of all the organisms present in the dis- 
charge and prepare a suspension which shall have a standardized content within 
the range of usefulness of the organisms present. Here we are confronted 
with the same proposition as in the case of mixed stock vaccines. All the organ- 
isms should be identified, if not they should not be used. In this way a more 
reliable and more scientific product will be furnished. 

2. Antityphoid Vaccination. 

The basis of the use of vaccines, as a preventive immunizing inoculation 
against typhoid fever, is the observation that one attack usually confers immunity 
from further attacks. This use of vaccines has become widespread and is 
promising great results, especially in those subjected to variations in the sani- 
tary surroundings. The doses are given at ten-day intervals; the first dose 
contains 500 million organisms and the second and third doses 1 billion bacteria 
each. A local reaction is usually present but this subsides within 48 hours, 
while a general systemic reaction is not always observed. As this measure 
is of such importance to health and sanitary officers, as well as to physicians in 
general, I give, herewith, some references for consultation. 1 

BIBLIOGRAPHY. 

I. Besson. Practical Bacteriology, Microbiology and Serum Therapy. 
London, 1913. 

1 See Wright, Lancet, 1896, II, 807; Brit. Med. Jour., 1897, I, 16; Leishman, Jour. Roy. 
Inst. Pub. Health, 1910, XVIII, 394; Firth, Jour. Roy. Army Med. Corps, 1911, XVII, 495; 
Russell, Boston Med. and Surg. Jour., 1911, CLXW, 1; Nelson and Hall, Jour. Am. Med. 
Assn., 1911, LVII, 1759; Schereschewsky, Pub. Health Rep., 1911, XXVI, 1508; Davis, Jour. 
Am. Med. Assn., 1912, LVIII, 537; Russell, Ibid., 1331; Maverick, Ibid., 1672; Engelbach, 
Interstate Med. Jour., 19 12, XIX, 537; Albert and Mendenhall, Am. Jour. Med. Sc, 1912, 
CXLIII, 232; Williams, Ibid., 352; Callison, Ibid., 1912, CXLIV, 350; RusseU, Bull. 2, War 
Dept. Office Surg. Gen., Washington, 1913, 7; Craig, Ibid., 15; Nichols, Ibid., 23; Hunt, Am. 
Jour. Med. Sc, 1913, CXLV, 826; Hiss, Jour. Med. Research, 1913, XXVIII, 385; Russell, 
Jour. Am. Med. Assn., 1913, LXI, 666; Metchnikoff and Besredka, Ann. de l'lnst. Pasteur, 
1913, XXVII, 597; Russell, Am. Jour. Med. Sc, 1913, CXLVI, 803 ; Foxworthy, Military Surg., 
1914, XXXIV, 332; Sachs. Med. Klin., 1914, X, 1538; Russell, Jour. Am. Med. Assn., 1914; 
LXII, 1371; Krumbhaar and Richardson, Am. Jour. Med. Sc, 1915, CXLIX, 406; Thomsen, 
Hospitals tid., 1915, LVIII, 211; Stursberg and Klose, Munch, med. Wchnschr., 1915, LXII, 
380; Cecil, Jour. Infect. Dis., 1915, XVI, 26; Biedl, Wien. klin. Wchnschr., 1915, XXVII, 125; 
Paltauf, Ibid., 125; Kirschbaum, Ibid., 208; Eggerth, Ibid., 209; Csernel, Marton and Feistman- 
tel, Ibid., 229; Sladek and St. Kotlowski, Ibid., 389; Goldscheider and Aust, Deutsch. med. 
Wchnschr., 1915, XLI, 361; Kisskalt, Ibid., 393; Schneider, Ibid., 393; Reibmayr, Munch, 
med. Wchnschr., 1915, LXII, 610; Rhein, Ibid., 427; Bourke, Evans and Rowland, Brit. Med. 
Jour., 191 5, 1, 584; Widal, Bull, de l'Acad. Med., 1915, LXXIV, 205; Vincent and Chantemesse, 
Ibid., 225; Harris and Ogan, Jour. Am. Med. Assn., 1915, LXFV, 3; Garbat, Ibid., 489; 
Trowbridge, Finkle and Barnard, Ibid., 728; Elmer, Ibid., 1147; Lyster, Ibid., 1915, LXV, 510; 
Jobling and Petersen, Ibid., 515; Sawyer, Ibid., 1413. 



CLINICAL BACTERIOLOGY. 727 

2. Hewlett. A manual of Bacteriology. St. Louis, 1914. 

3. Irons. Billings-Forchheimer Therapeusis, vol. 5. New York, 19 14. 

4. Jordan. General Bacteriology. Philadelphia, 1913. 

5. Kolle and Hetsch. Die Experimentelle Bakteriologie und die Infektions- 

krankheiten. Berlin, 1911. 

6. Kolle and Wassermann. Handbuch der Pathogenen Mikroorganismen. 

Jena, 1912 and 1913. 

7. Kraus and Levaditi. Handbuch der Technik und Methodik der Immuni- 

tatsforschung. Jena, 1909-1913. 

8. McFarland. Text-book on Pathogenic Bacteria. Philadelphia, 1913. 

9. Park and Williams. Pathogenic Microorganisms. Philadelphia, 19 14. 



General Clinical Diagnosis. 

1. Boston. A Text-book of Clinical Diagnosis. Philadelphia, 1905. 

2. Brown and Ritchie. Medical Diagnosis. Edinburgh, 1906. 

3. Brugsch and Schittenhelm. Lehrbuchklinischer Untersuchungsmethoden 

fur Studierende und Aerzte. Berlin, 1911. Technik der Speziellen 
Klinischen Untersuchungsmethoden, Berlin, 19 14. 

4. Butler. Diagnostics of Internal Medicine. New York, 1905. 

5. Da Costa. Medical Diagnosis. Philadelphia, 1890. 

6. Debove et Achard. Manuel de diagnostic medical. Paris, 1900. 

7. Deguy et Guillaumin. Microscopie clinique. Paris, 1912. 

8. EMERson. Clinical Diagnosis. Philadelphia, 19 13. 

9. French. Medical Laboratory Methods and Tests. Chicago, 1912. 

10. Guiart et Grimbert. Precis de diagnostic. Paris, 191 2. 

11. Greene. Medical Diagnosis. Philadelphia, 1907. 

12. Hare. Practical Diagnosis. Philadelphia, 1908. 

13. Hutchinson and Rainy. Clinical Methods. Chicago, 1908. 

14. Jagic. Klinische Mikroskopie. Wien, 1913. 

15. Koranyi und Richter. Physikalische Chemie und Medizin. Leipzig, 1908. 

16. Von Jaksch. Klinische Diagnostik. Berlin, 1910. 

17. Klemperer. Grundriss der klinischen Diagnostik. Berlin, 1906. 

18. Kraft. Analyt. Diagnostikum. Leipzig, 1913. 

19. Lenhartz. Mikroskopie und Chemie am Krankenbett. Berlin, 1913. 
20.. Leube. Specielle Diagnose der inneren Krankheiten. Leipzig, 1908. 

21. Morris. Clinical Laboratory Methods. London, 19 13. 

22. Musser. Medical Diagnosis. Philadelphia, 1904. 

23. Von Noorden. Handbuch der Pathologie des Stoflwechsels. Berlin, 1906. 
24.. Panton. Clinical Pathology. London, 19 13. 

25. Sahli. Lehrbuch der klinischen Untersuchungsmethoden. Wien, 1913. 

26. Schilling. Aerztliche Technik. Wiirzburg, 1906. 

27. Simon. A Manual of Clinical Diagnosis. Philadelphia, 1914. 

28. Todd. Manual of Clinical Diagnosis. Philadelphia, 191 2. 

29. Wesener. Medizinisch-klinisch Diagnostik. Berlin, 1907. 

30. Wood. Chemical and Microscopical Diagnosis. New York, 19 11. 

31. Zuelzer. Chemische und Mikroskopische Diagnostik. Leipzig, 1906. 



INDEX 



Abderhalden's sero-diagnosis of cancer, 
650 

of dementia praecox, 651 

of pregnancy, 638 
Abortion, 384 
Abscess, blood in, 546 

indican in, 257 

of liver, sputum in, 33 

of lung, sputum in, 33 
Absorptive power of stomach, 86 
Acanthia lectularia, 164 
Acarus scabiei, 162 
Accidental albuminuria, 261 
Acetic acid in gastric contents, 64, 73, 

77 
Aceto-acetic acid, 191, 324 
Acetone bodies, 191, 317 

in blood, 451 

in urine, 191, 317 

determination of, 322 
significance of, 317 
tests for, 320 
Acetonemia, 451 
Acetonuria, 317 
Acholic stools, 107, 116 
Achorion Schonleinii, 165 
Achroiocythemia, 426 
Achromatophilia, 496 
Achroodextrin, 36, 82 
Achylia gastrica, 78, 89 
Acid, acetic, 64, 73, 77, 249 

aceto-acetic, 191, 324 

alloxyproteic, 208, 248 

amino, 82, 191, 246 

bile, 61, 109, 191, 331 

butyric, 64, 73, 7j, 249 

cholalic, 117, 191 

chondroitin-sulphuric, 191, 208, 

253 
diacetic, 191, 324 
diamino, 83, 92, 246 
fatty, 13, 115, 123, 191, 249, 449 
formic, 249 
glycocholic, 117, 191 
glycosuric, 333 
glycuronic, 191, 314 
hippuric, 191, 248, 345 
homogentisic, 191, 333 
hydrochloric, 51, 65 
hydroquinone-acetic, 333 
lactic, 64, 73, 74, 249 
nucleinic, 232, 253 
oxalic, 191, 250 
oxaluric, 191, 250 
/?-oxybutyric, 191, 325 
oxymandelic, 191 
oxyproteic, 191, 208, 248 
phosphoric, 191, 198 



Acid, picric, 272 

propionic, 249 

rosacic, 254 

rosolic, 21 

sulphuric, 206 

taurocholic, 117, 191, 331 

uric, 191, 232, 439 

uroferric, 208 

uroleucic, 191, 333 
Acid-fast organisms, 20 
Acidity of gastric juice, 64 

of urine, 184, 186 
Acidophilic cells, 496 

granules, 499, 506 
Acidosis, 227, 317 
Acid stains, 477 

unit, 186 
Actinomyces in sputum, 27 
Actinomycosis, 27 
Acute anterior poliomyelitis, 684 

bronchitis, sputum in, 31 

gastritis, gastric juice in, 90 

hemorrhage, anemia due to, 535 

infectious conjunctivitis, 47 
diseases, blood in, 549 

leukemia, 543 

nephritis, 178, 181, 190, 194, 201, 
217, 265 

rheumatism, blood in, 557 

yellow atrophy of the liver, urine 
in, 220, 246, 343 
Addison's disease, blood in, 548 
Adenin, 240 

Adler's test for blood, 1 10 
Adolescent albuminuria, 264 
Aerobic cultures, 706 

organisms, 706 
^Estivo- autumnal malaria, 566 
Agar, ameba, 703 

ascitic, 704 

blood, 703 

dextrose, 703 

glycerin, 703 

hydrocele, 704 

litmus lactose, 703 

nutrient, 702 
Age, effect of, on red blood cells, 493 

on white blood cells, 521 
Agglutination, 598, 607 
Agglutinins, 598 
Agglutinophore, 598 
Agonal leucocytosis, 516 
Air in sputum, 4 
Albumin, determination of, 272 

in exudates, 664 

in feces, 122 

in milk, 693 

in sputum, 5 



729 



73° 



INDEX. 



Albumin, in transudates, 663 

in urine, 261 ' 

of blood, 428 

quotient, 275, 429 

removal of, 274 

serum, 261 

significance of, in urine, 261 

tests for, 266 
Albuminuria, accidental, 261 

adolescent, 264 

after baths, 262 

alimentary, 262 

colliquative, 265 

constitutional, 263 

cyclic, 263 

false, 261 

febrile, 264 

functional, 261 

hematogenous, 265 

hypostatic, 264 

intermittent, 263 

lordotic, 263 

mixed, 280 

neurotic, 265 

of the new-born, 262 

of pregnancy, 262 

orthostatic, 263 

orthotic, 263 

physiologic, 261 

post-infectious, 264 

postural, 263 

renal, 265 

structural, 265 

thermolytic, 278 

toxic, 265 

traumatic, 265 

true, 261 

vesicular, 261 

with definite renal lesions, 265 
Albumon, 427 
Albumose, Bence-Jones, 276 

in blood, 429 

in feces, 122 

in gastric contents, 82 

in urine, 276 
Albumosuria, 276 

alimentary, 280 

digestive, 280 

enterogenous, 280 

febrile, 280 

hematogenous, 280 

-hepatogenous, 280 

myelopathic, 277 

pyogenic, 280 

renal, 280 

significance of, 279 

tests for, 279 
Alcohol-fast organisms, 20 
Aleukemic myelosis, 544 
Alimentary albuminuria, 262 

albumosuria, 280 

chloruria, 193, 372 

galactosuria, 305, 312 

glycosuria, 285 

levulosuria, 305 

lipuria, 346 

pentosuria, 308 
Alkaline phosphates, 199 

tide of urine, 186 



Alkalinity of blood, 395 
Alkalinuria, 200 
Alkaptonuria, 333 
Allantoin, 191, 248 
Allergy, 600 

Alloxur bases, 122, 191, 240, 341 
Alloxyproteic acid, 191, 208, 248 
Almen-Nylander's test for glucose, 292 
Almen's tannic acid solution, 692 
Aloin test for blood, 109, 655 
Altitude, effect of, on red cells, 493 
Alveolar epithelial cells in sputum, 1 1 
Ambard's coefficient, 438 
Amblyochromatic erythroblasts, 491 
Amboceptors, 618, 624, 625, 630 
Amboceptor paper, 630 
Ameba agar, 703 
Ameba coli in feces, 134 

in mouth, 42 

in sputum, 28 

in urine, 366 

pulmonalis, 28 
Amebic dysentery, 135 

pyorrhea, 42 
American hook-worm, 152 
Amino-acetic acid, 246 
Amino acids in urine, 191, 246 
a-aminoisobutyl-acetic acid, 343 
Ammonia in blood, 443 

in urine, 227 
Ammoniemia, 443 

Ammonium magnesium phosphate in 
feces, 120 
in sputum, 13 
in urine, 348 

urate calculi, 368 
sediment, 347 
Amniotic fluid, 383 
Amount of blood, 387 

of cerebrospinal fluid, 680 

of feces, 104 

of gastric juice, 57 

of sputum, 2 

of urine, 177 
Amyloid kidney, globulin in urine of, 

275 
urine in, 178, 182, 253, 266, 275 
Amylopsin, 102 
Amylosis, 34 
Anachlorhydria, 70 
Anaerobic cultures, 706 

organisms, 706 
Anaphylaxis, 723 
Ancylostoma duodenale, 151 
Ancylostomiasis, 151 
Anemia, 528 

aplastic, 534 
Biermer's, 531 
chlorotic, 529 
definition of, 528 
Ehrlich's, 534 

due to acute hemorrhage, 535 
to acute infections, 538 
to bad air, 537 
to blood poisons, 538 
to chronic diseases, 557 
to chronic hemorrhage, 536 
to inanition, 536 
to intestinal parasites, 152, 537 



37 



INDEX. 



731 



Anemia, febrile, 538 

hemolytic, 538 

infantum pseudoleukemica, 533 

von Jaksch's, 533 

leukanemia, 534 

lymphatic, 543 

of the South, 152 

of the tropics, 493 

primary pernicious, 531 

progressive pernicious, 531 

secondary, 534 

simple primary, 528 

splenic, 532 
Anemic degeneration, 497 
Anesthesia, changes in urine after, 

319 

effects of, on blood, 547 
Angioneurotic hematuria, 355 
Anguillula aceti in urine, 366 

intestinalis et stercoralis, 147 
Anhydremia, 390 
Animal gum in urine, 314 
Animal parasites in blood, 561 

in ear, 47 

in feces, 133 

in gastric contents, 63 

in sputum, 28 

in urine, 366 
Ankylostomum duodenale, 151 
Anopheles maculipennis, 561 
Anterior urethritis, 353 
Anthracosis, 4, 34 
Antiamboceptors, 597 
Antibodies, 595, 619 
Anticomplement, 597 
Antiformin method for tubercle bacilli, 

18 
Antigens, 595, 619, 620, 622, 626 
Antihemolysins, 597 
Antihuman amboceptor, 630 
Antimeningococcic serum, 683 
Antisheep amboceptor, 624 
Antitoxins, 594 

Antityphoid vaccination, 600, 726 
Anuria, 180 
Aplastic anemia, 534 
Appearance of blood, 393 

of exudates, 664 

of feces, 105 

of gastric contents, 57 

of leucocytes, 502 

of milk, 690 

of red cells, 486 

of semen, 376 

of spinal fluid, 680 

of sputum, 4 

of transudates, 663 

of urine, 180 
Appetite juice, 51 
Arabinose, 309 
Arginin, 247 
Arneth's classification of neutrophiles, 

505 
Arnold's test for diacetic acid, 325 
Arnold- Volhard m ethod for chlorids, 195 
Arterial blood, 394 
Arthropoda, 161 
Ascaridae in feces, 145 
Ascaris alata, 146 



Ascaris caniculae, 146 

canis, 146 

canis et martis, 160 

cati, 146 

felis, 146 

graecorum, 147 

lumbricoid.es, 145 

lumbricus canis, 146 

marginata, 146 

my st ax, 146 

teres, 146 

trichiura, 149 

tricuspidata, 146 

vermicularis, 147 

visceralis et renalis, 160 

werneri, 146 
Ascitic agar, 704 

fluid, cytology of, 676 
Asexual cycle of malarial parasite, 564 
Ash-free diet of Taylor, 195 
Asiatic cholera, feces in, 106, 129 

organism of, 129 
Aspergillus flavus, 16 

fumigatus, 16 

in aural secretion, 47 

in sputum, 16 

niger, 16 

subfuscus, 16 
Assimilation limit, 284 
Asthma, bronchial, 33 

eosinophilia in, 10, 33, 519 

sputum in, 33 
Atrophic gastritis, 92 
Aural secretion, 46 

bacteria in, 46 

larvae in, 47 

molds in, 47 
Autotoxic enterogenous cyanosis, 496 
Autovaccines, 600, 725 
Azoospermatism, 378 
Azotorrhea, 114 

Babcock's method for fat in milk, 693 
Bacillary dysentery, 132 

index, 599 
Bacilluria, 366 
Bacillus aerogenes capsulatus, 1 33 

anthracis, 26 

coli communis, 48, 129, 366, 589, 712 

comma, 61, 129, 715 

Ducrey's, 669 

hodgkini, 544, 711 

icteroides, 583 

lepras, 24 

mallei, 27 

mucosus capsulatus, 25, 46 

of Boas-Oppler, 62 

of Bordet-Gengou, 26, 711 

of bubonic plague, 27 

of diphtheria, 38, 709 

of dysentery, 132, 713 

of Finkler-Prior, 61, 130 

of Friedlander, 25, 46 

of glanders, 27 

of Hansen, 24 

of Hoffman, 710 

of influenza, 26, 711 

of Kitasato and Yersin, 27 

of Klebs-Loffler, 38, 709 



732 



INDEX. 



Bacillus of Koch, 18, 132, 364, 592, 667 

of Koch-Weeks, 48 

of Morax-Axenfeld, 48 

of ozena, 46 

of Perez, 46 

of Pfeiffer, 26, 33, 711 

of Sanarelli, 583 

of Shiga, 132,713 

of soft chancre, 669 

of tuberculosis, 17, 132, 364, 592, 
667 

of Tunnicliff, 45 

of Vincent, 40 

of Welch, 133 

of' whooping-cough, 26, 7 1 1 

paratyphoid, 131, 588 

pertussis, 26, 711 

pestis, 27 

pseudo-diphtheria, 40, 710 

pyocyaneus, 4 

rhinitis, 45 

smegma, 24, 364, 669 

timothy, 24 

typhosus, 26,130,366,587,712 

ulceris cancrosi, 669 

xerosis, 40 

X of Sternberg, 583 
Bacteria in blood, 585 

in conjunctiva, 47 

in ear, 46 

in exudates, 667 

in feces, 128 

in gastric contents, 62 

in milk, 696 

in mouth, 36 

in nasal secretions, 44 

in sputum, 14 

in urine, 363 
Bacterial flora of feces, 129 

of vagina, 380 
Bacterial vaccines, 598, 723 
Bacteriemia, 585 
Bacteriology of blood, 585 

of cerebrospinal fluid, 682 

of exudates, 667 

of feces, 128 

of milk, 696 

of sputum, 14 

of urine, 363 
Bacteriolysins, 595 
Bacteriuria, 363 
Balantidium coli, 138 
Baldwin's method for oxalic acid, 251 
Bang's test for albumose, 279 

for sugar, 296 
Banti's disease, blood in, 532 
Barberio's test for semen, 379 
Barber's itch, organism of, 166 
Bases, alloxur, 122, 191, 240, 341 

hexone, 83, 92, 247 

nuclein, 122, 191, 240, 341 

purin, 122, 191, 240, 341 

xanthin, 122, 191, 240, 341 
Basic stains, 477 
Basket cells, 509 
Basophile leucocytes, 506 
Basophiles, 506 
Basophilia, 498, 506 
Basophilic degeneration of red cells, 498 



Basophilic stippling of reds, 498 
Bass and Watkin's macroscopic agglu- 
tination test, 611 
Baths, albuminuria following, 262 

effect of, on red cells, 494 
Beckmann apparatus, 405 
Bed bug, 164 
Beef tape- worm, 141 
Bence-Jones, body, 276 
protein, 276 

amount of, 276 
significance of, 276 
tests for, 277 
Benedict and Murlin's test for amino 

acids in urine, 247 
Benedict's test for sugar, 290 
Benzidin test for blood, no 
Benzoic acid, 248 
Bial's test for pentose, 310 
Biermer's anemia, 531 
Bile acids in blood, 451 
in feces, 117 
in gastric contents, 61 
in urine, 191, 331 
media, 713 

pigments in blood, 451 
in exudates, 664 
in feces, 108, 117 
in gastric contents, 59, 61 
in sputum, 3 
in urine, 191, 329 
significance of, 329 
tests for, 329 
Bilharzia hematobia, 29, 585 
Bilharziasis, 585 
Bilicyanin, 117 
Bilifuscin, 117, 329 
Bilihumin, 117 
Biliprasin, 108, 329 
Bilirubin, 59, 108, 117. 329, 345 
Biliverdin, 59, 108, 117, 329 
Biologic test for blood, 614 
Bismuth oxid in stools, 107 

test for glucose, 292 
Biuret test for protein, 279 
Black's method for ,3-oxybutyric acid, 

326 
Black sputum, 4 
urine, 182 
-water fever, 281 
Bladder, inflammation of, 187, 352 

tuberculosis of, 365 
Blastomycetes in skin, 169 

in sputum, 17 
Blastomycosis, 17, 169 
Blennorrhea, 381 
Blood, 385 

acetone in, 451 

after anesthesia, 546 

after splenectomy, 560 

after surgical intervention, 546 

agar, 703 

albumin in, 428 

alkalinity of, 395 

amino acids in, 445 

ammonia in, 443 

bacteriology of, 585 

biliary constituents in, 451 

carbohydrates in, 445 



INDEX. 



733 



Blood, casts, 359 
cells in, 486 

chemical properties of, 407 
chemical tests for, 109, 655 
cholesterin in, 450 
coagulation of, 401 
color-index of, 426 
color of, 394 
constituents of, 394 
counting of cells of,- 455, 468, 510 
creatinin in, 444 
crises, 491 
cryoscopy of, 405 
cultures, 586 
dust, 525 

electric conductivity of, 406 
enumeration of cells of, 455, 460, 

468, 505, 510 
fat in, 449 
ferments of, 454 • 
fixation of smears of, 473 
formation of, 386 
fresh, 470 
gases in, 454 
glucose in, 446 
hemoglobin of, 409 
in abscess formation, 546 
in acute infections, 549 
in acute rheumatism, 557 
in Addison's disease, 548 
in aplastic anemia, 534 
in bilharziasis, 585 
in carcinoma, 559 
in chlorosis, 529 
in chronic diseases, 557 
in chronic tuberculosis, 557 
in diabetes mellitus, 547 
in diphtheria, 556 
in distomiasis, 585 
in filariasis, 577 
in gout, 548 
in kala-azar, 576 
in leprosy, 559 
in leukanemia, 534 
in leukemia, 539 
in malaria, 561 
in measles, 554 
in myxedema, 549 
inorganic constituents of, 452 
in pernicious anemia, 531 
in pertussis, 556 
in pneumonia, 550 
In primary anemia, 528 
in pseudoleukemia, 544 
in relapsing fever, 574 
in rickets, 549 
in Rocky Mountain spotted fever, 

584 
in scarlet fever, 553 
in secondary anemia, 534 
in sleeping sickness, 575 
in splenic anemia, 532 
in syphilis, 558, 578 
in typhoid fever, 552, 587 
in varicella, 555 
in variola, 555 
in whooping-cough, 556 
in yellow fever, 583 
limitations of examinations of, 659 



Blood, medico-legal aspects of, 654 
tests for, 654 
morphology of, 469 
needle, 392 
nitrogen of, 430 
non-protein nitrogen of, 430 
obtaining of, 392 
odor of, 394 

osmotic pressure of, 405 
parasitology of, 561 
pathology of, 528 
general, 546 
special, 528 
physiology of, 386 
pigments of, 409 
plates, 468, 523, 526 
properties of, 393 
proteins of, 427 
reaction of, 395 
red cells of, 486 
serum reactions of, 606 
smears of, 470 
solids of, 409 
specific gravity of, 399 
spectroscopic tests for, 410, 658 
staining of smears of, 476 
tests for, 109, 654 
total solids of, 409 
urea in, 435 
uric acid in, 439 
value of examinations of, 659 
viscosity of, 401 
vital staining of, 485 
volume of, 387 
volume relations of elements of, 

white cells of, 502 
Blood casts, 359 

cells in exudates, 665 
in feces, 120 
in gastric contents, 61 
in sputum, 3, 5, 11 
in urine, 328, 354 
plates, 523 

appearance of, 523 
counting of, 468 
function of, 525 
number of, 524 
size of, 524 

staining properties of, 524 
poisons, 538 
smears, fixation of, 473 
preparation of, 470 
staining of, 476 
staining, 476 
Bloody sputum, 3, 5, 11 
Boas' method for lactic acid, 76 

for estimating gastric motility, 85 
test for free hydrochloric acid, 67 
test-meal, 55 
Boas-Oppler bacillus, 62 
Body louse, 163 
Boggs' coagulometer, 403 

method for protein in milk, 692 
Bone-marrow, 386, 525 
function of, 386 
morphology of, 525 
Borax as preservative in milk, 696 
Bordet's potato medium, 711 



734 



INDEX. 



Boric acid as preservative, 696 
Bothriocephaloidea in feces, 144 

bothriocephalus latissimus, 144 

bothriocephalus latus, 144 

bothriocephalus sp. Ijima et Kuri- 
moto, 145 

dibothriocephalus cordatus, 144 
Bottcher's crystals, 14, 376 
Bouillon, 701 
Bradshaw's myelopathic albumosuria, 

277 
Breakfast, test, 54 
Bremer's blood-test in diabetes, 547 
Bricklayers' anemia, 153 
Brodie-Russell coagulometer, 403 
Bronchial asthma, 33 

stones, 8 
Bronchioliths, 8 
Bronchiolitis exudativa, 7 
Bronchitis, acute, 31 

chronic, 32 

eosinophilic, 10 

fetid, 32 

fibrinous, 32 

putrid, 32 
Broncho-pneumonia, sputum of, 31 
Bronfenbrenner's modification of Abder- 

halden test, 647 
Bunge-Trantenroth's method for tuber- 
cle bacilli, 24 
Burri's method for spirochete, 670 
Busk's intestinal fluke, 158 
Butyric acid in gastric contents, 64, 73, 
77 

Cabot's ring bodies, 499 
Cachexial fever, 576 
Cadaverin, 123 
Caffein, 240 

Calcium carbonate calculi, 370 
sediment, 349 

of urine, 2 1 1 

oxalate calculi, 368 
crystals in sputum, 14 
in urine, 250, 341 

phosphate calculi, 368 
sediment, 346, 347 

soaps in feces, 120 

sulphate sediment, 345 
Calculi, ammonium urate, 368 

biliary, 117 

bronchial, 8 

calcium carbonate, 370 
oxalate, 368 

classification of, 367 

cystin, 370 

examination of, 369 

formation of, 367 

hepatic, 117 

intestinal, 118 

nasal, 46 

phosphatic, 368 

pulmonary, 8 

renal, 367 

table for examination of, 369 

ureteral, 367 

urethral, 367 

uric acid, 368 

urostealith, 370 



Calculi, vesical, 367 

xanthin, 370 
Calmette's tuberculin reaction, 602 
Cammidge's reaction, 311 
Cancer (see Carcinoma) 
Cane sugar, absorption of, 123 

digestion of, 64 

in urine, 314 
Capsule, staining of, 590 
Carbohydrates, digestion of, 36, 64, 82 

in blood, 445 

in exudates, 664 

in feces, 116, 123 

in milk, 695 

in urine, 283 
Carbol-fuchsin solution, 20 
Carbonates in urine, 211 
Carbon dioxid hemoglobin, 412 
in blood, 454 

monoxid hemoglobin, 412 
poisoning, 412 
Carcinoma, Abderhalden's test in, 650 

blood in, 559 

cells in exudates, 675 

fragments in feces, 118 
in gastric contents, 63, 93 
in urine, 363 

of cervix, 384 

of kidney, 354, 363 

of pleura, 675 

of rectum, 118 

of stomach, 91 

of uterus, 384 
Cardiac albuminuria, 266 

edema, 179 

pleurisy, 675 
Carnin, 240 
Casein in feces, 115 

appearance of, 115 
Leiner's test for, 115 

in milk, 693 
Casts, 356 

blood, 359 

chemistry of, 356 

colloid, 360 

epithelial, 359 

fatty, 359 

fibrinous, 359 
in sputum, 8 

granular, 357 

hyaline, 356 

mixed, 356 

origin of, 356 

prostatic, 378 

pseudo, 361 

pus, 359 

significance of, 357, 361 

size of, 356 

staining of, 357 

testicular, 378 

true, 356 

waxy, 358 
Catalase, 522 
Catarrhal stomatitis, 38 
Cellulose in feces, 117 
Centrifugation, 338 
Cercomonads in feces, 138 

in sputum, 28 

in urine, 366 






INDEX. 



735 



Cercomonads in vaginal secretions, 381 
Cercomonas coli hominis, 137 

hominis, 138 

intestinalis, 138 

sett Bodo urinarius, 137 
Cerebrospinal fluid, 678 

bacteriology of, 682 

chemistry of, 681 

cytology of, 682 

in nasal secretion, 45 

microscopy of, 682 

obtaining of, 679 

pressure of, 679 

properties of, 680 

syphilis, 685 
Cerumen, 46 
Cestodes in feces, 139 

in sputum, 29 
Chalicosis, 4, 34 
Chancre, organism of, 669 
Character of blood, 393 

of exudates, 664 

of feces, 105 

of sputum, 4 

of urine, 176 
Charcoal in feces, 101 
Charcot- Leyden crystals in feces, 120 

in sputum, 14, 30, 31 
Cheesy masses in sputum, 6 
Chemical fixation of smears, 475 
Chemotaxis, 598 
Childhood, red cells in, 500 

white cells in, 521 
Chinese liver-fluke, 159 
Chinovose, 308 
Chloremia, 426 
Chlorid excretion in urine, 192, 372 

retention, 192 
Chlorids of the blood, 452 

of the urine, 192 
amount of, 192 
estimation of, 195 
variations of, 192 
Chloromata, 4 
Chlorosis, 529 
Chlorotic anemia, 529 
Chloruria, 192, 372 
Cholecyanin in feces, 107 

in urine, 329 
Cholelithiasis, 117 
Cholemia, 451 
Cholera red reaction, 716 

spirillum, characteristics of, 715 
in feces, 129 
Cholesterin blood, 450 

crystal s in feces, 120 

in sput u m, 13 

in urine, 346 
Choletelin in urine, 329 
Choluria, 329 
Chondroitin-sulphuric acid, 191, 208, 

253 

Chromogenic bacteria in sputum, 4 
Chromogens in urine, 254 
Chronic bronchitis, sputum in, 32 
diseases, blood in, 557 
gastritis, gastric juice in, 90 
nephritis, urine in, 178, 181, 190, 
194, 201, 266 



Chyloid exudates, 665 
Chylous exudates, 665 
Chyluria, 182, 346 
Chymosin, 64, 81 
Cimsenomonas hominis, 138 
Cimex lectularius, 164 
Cladoccelium hepaticum, 157 
Clark's method for elastic tissue, 12 
Clay-colored stools, 107, 117 
Cleaning glass-ware, 469 
Clearing of urine, 298, 300 
Coagulation of blood, 401 

of exudates, 664 

of milk, 691 

of urine, 283 

time of blood, 403 
Coagulometer of Boggs, 403 

of Dorrance, 402 

of Rudolf, 403 

of Russell- Brodie, 403 
Coal pigment in sputum, 4, 34 
Coarsely granular cells of Schultze, 506 
Coating of the tongue, 38 
Coccidioidal granuloma, 170 
Coccidioides immitis, 170 
Coccidium hominis, 137 

perforans, 137 
Coccobacillus fcetidus ozenae, 46 
Coefficient, creatinin, 242 

of Ambard, 438 

of Haeser, 189 

of Haines, 189 

of Long, 189 

refraction, 389, 429 
Colitis, catarrhal, 116 

malignant, 118 

mucous, 112 
Collection of feces, 99 

of gastric contents, 52 

of puncture fluids, 663 

of sputum, 2 

of urine, 176 
Collignon and Pilod's test, 683 
Colloid casts, 360 

cysts, 676 
Colloidal nitrogen, 248 
Colon bacillus, 48, 129, 366, 589, 712 
Color-index of blood cells, 426 

of blood, 394 

of exudates, 664 

of feces, 106 

of gastric contents, 58 

of sputum, 3 

of urine, 181 
Colorimeter, 244 
Colostrum, 688 
Coma diabeticum, 318 
Combined hydrochloric acid, 71 
Comma bacillus of Koch, 61, 129, 715 
Common flea, 165 

liver-fluke, 157 
Complement, 597, 618, 624, 625 

fixation test, 617, 636J 
Complementophile, 597 
Composition of blood, 407 

of milk, 689 

of urine, 190 
Concretions, biliary, 117 

in sputum, 8 



736 



INDEX. 



Concretions in bronchioliths, 8 

in pneumoliths, 8 

intestinal, 118 u 

coproliths, 118 
enteroliths, 118 

nasal, 46 

renal, 367 

vesical, 367 
Conductivity, electric, of blood, 406 

of urine, 371 
Congo-red test, 66 

Conjugated glycuronic acids, 124, 314 
Conjunctival secretions, 47 
Conjunctivitis, diphtheritic, 47 

gonorrheal, 48 

infectious, 47 

vernal, 49 
Consistency of blood, 401 

of feces, 105 

of gastric contents, 59 

of milk, 689 

of spinal fluid, 680 

of sputum, 2 

of urine, 181 
Constipation, 105 
Coproliths, 118 
Corpora amylacea, 377 
Cory neb acterium granulomatis ma- 

ligni, 711 
Cough, whooping, blood in, 556 

organism of, 26, 711 
Counting of blood plates, 468 

of pus cells, 354 

of red cells, 455 

of white cells, 465 
Crab louse, 163 
Creatin in urine, 241, 245 
Creatinin coefficient, 242 

estimation of, 243 

metabolism of, 241 

tests for, 243 

variations of, 241 
Creatorrhcea, 114 
Crenation, 487 
Crescents in blood, 567 
Crises, blood, 491 
Cryoscopy of blood, 405 

of urine, 371 
Crystals in feces, 120 

in gastric contents, 63 

in semen, 376 

in sputum, 13 

in urine, 338 
Culex mosquito, 583 
Cultures, aerobic, 706 

anaerobic, 706 

blood, 586 

media, 701 

throat, 39 

urine 363 
Curds in feces, 115 
Curschmann's spirals, 7, 30, 31 
Cutaneous reactions, 600, 603 
Cyclic albuminuria, 263 
Cylindroids, 360 
Cylindruria, 361 
Cyst, colloid, 676 

dermoid, 677 

fluids, 676 



Cyst,' hydatid, 678 

hydrocele, 677 

hydronephrotic, 678 

myxoid, 676 

ovarian, 676 

pancreatic, 678 

papillary, 677 

parovarian, 677 

serous, 676 

spermatocele, 678 
Cystein, 208 

Cysticercus cellulosae, 141 
Cystin, 208 

calculi, 370 

sediment, 342 
Cystinuria, 342 
Cystitis, 187, 352, 365 
Cystospermium hominis, 137 
Cystotaenia solium, 140 
Cytology in cardiac pleurisy, 676 

in malignant pleurisy, 676 

in nephritic pleurisy, 676 

in pneumococcus pleurisy, 674 

in primary tubercular pleurisy, 
674 

in secondary tubercular pleurisy, 
674 

in streptococcus pleurisy, 675 

in typhoid pleurisy, 675 

of ascitic fluid, 676 

of cerebrospinal fluid, 682 

of exudates, 672 
of peritoneum, 675 
of pleura, 674 

of normal-fluids, 673 

of sputum, 10 

technic of, 672 
Cytophile, 597 

Dahlia stain, 507 
Daland's hematocrit, 390 
Dare's hemoalkalimeter, 396 

hemoglobinometer, 420 

method for alkalinity of blood, 396 
Darling's method of staining amebae, 

• I36 
Day urine, 178 

Dechloridization, 194 

Deficit of hydrochloric acid, 73 

Definitive host, 140 

Degenerated forms of red ceils, 498 

of white cells, 509 
Degeneration, anemic, 497 

hemoglobinemic, 499 
Degree of tolerance, 284 
Delayed chloroform poisoning, 319 
Dementia praecox, Abderhalden's test 

in, 651 
Demodex folliculorum, 162 
Dermacentor Andersoni, 584 

venustus, 584 
Dermoid cysts, 677 
Desmoid bag, 87 
Deutero-albumose, 82, 276 
Dextrin in urine, 314 
Dextrose agar, 703 
Diabetes alternans, 234 

insipidus, 179 

mellitus, 179, 182 



INDEX. 



737 



Diabetes mellitus, blood in, 547 

Bremer's test in, 547 

lipemia in, 547 

urine in, 179, 182 

Williamson's test in, 548 
phosphatic, 201 
renal, 284 
Diabetic coma, 318 
Diacanthos polycephalus, 153 
Diacetic acid in urine, 324 
Diagnosis, functional, 370 
Diagnostic value of agglutination test, 

613 

of complement fixation test, 633 

of precipitin test, 616 
Diamines in urine, 123, 342 
Diamino acids, 83, 92, 246 
Diaminuria, 342 
Diarrhea, 105 
Diastase in urine, 251 
Diazo reaction, 334 
Dibothriocephalus cordatus, 144 

latus, 144 
Dibothrium latum, 144 
Diet of Folin, 101 

of Schmidt and Strasburger, 100 

of Taylor, 195 
Dieudonne's medium, 716 
Differential counting, 510 
Diffusible alkalinity of blood, 395 
Digestion, gastric, 82 

intestinal, 103, 121 

leucocytosis of, 513 

products of, 82, 103 
Digestive insufficiency, 113 
Dilatation of stomach, 83, 84 
Diluting fluids for blood, 459, 466 
Dimethylaminoazobenzol test, 66 
Dimethylaminobenzaldehyd reaction, 

336 
Dimorphous muris, 138 
Diphtheria, bacillus of, 38, 709 

blood in, 556 

taking smear in, 39 
Diphtheritic conjunctivitis, 47 

laryngitis, 39 
Diplacanthus nana, 142 
Diplococcus cinereus, 720 

crassus, 720, 722 

flavus, 720, 722 

intracellular! s meningitidis, 682, 722 

lanceolatus, 25, 31 

of Bonome, 663 

of Fraenkel, 25, 31, 589, 717 

of Jaeger and Heubner, 683 

of Neisser, 667, 720 

of Weichselbaum, 682, 722 

pneumoniae, 25, 31, 589, 717 
Diplogonoporus grandis, 145 
Diplomellituria, 287 
Dipylidium caninum, 141 

cucumerinum, 141 
Distoma capense, 585 

pulmonale, 29 

Ringeri, 29 

Westermanii, 29 
Distomiasis, 585 
Distomum buski, 158 

cavias, 147 

47 



Distomum conus, 159 

crassum, 158 

hematobium, 30, 585 

hepaticum, 157 

hepatis endemicum seu pernicio- 
sum, 159 

hepatis innocuum, 159 

japonicum, 159 

lanceolatum, 159 

sibiricum, 159 

sinense, 159 

spathulatum, 159 

tenuicolle, 159 
Ditrachyceros rudis, 153 
Dittrich's plugs, 6 
Dochmius ancylostomum, 151 

duodenalis, 151 
Dohle's inclusion bodies, 554 
Donne's test for pus, 354 
Donogany's test for hemoglobin, 282 
Doremus ureometer, 222 
Dorrance's method for coagulation- 
time, 402 
Drigalski and Conradi's media, 130 
Drop method for Wassermann test, 629 
Dropsical cells, 489, 529 
Dropsy of chorionic villi, 384 
Drugs, effects of, on blood, 429, 516 

reactions of, in urine, 183 
Dry test of feces for blood, in 
Ducrey's bacillus, 669 
Durham's hemocytometer, 467 
Dum-dum fever, 576 
Dwarf tape-worm, 142 
Dysentery, amebic, 135 

bacillary, 132 
Dysmenorrhea, 383 
Dyspepsia, 91 

Earthy phosphates, 199, 201 
Eberth's bacillus, 26, 130, 366, 587, 712 
Echinococcus in feces, 145 

in sputum, 9 

in urine, 366 
Ectasis, gastric, 85 
Eel, vinegar, 366 
Effusions, pleuritic, 674 
Egg-yellow reaction, 335 
Egyptian chlorosis, 153 
Ehrlich's anemia, 534 

anemic degeneration, 497 

classification of leucocytes, 510 

dahlia stain, 507 

diazo reaction, 334 

dimethylaminobenzaldehyd reac- 
tion, 336 

egg-yellow reaction, 335 

hemoglobinemic degeneration, 499 

side-chain theory, 594 

tri-acid stain, 480 

triple stain, 480 
Einhorn's method for total acidity, 72 

saccharometer, 303 
Elastic tissue in feces, 115 

in sputum, 12 
Electric conductivity of blood, 406 

of urine, 371 
Empyema, perforating, 34 
Endamceba buccalis, 42 



738 



INDEX. 



Endamceba coli, 137 

gingivalis, 42 

histolytica, 134 

tetragena, 135 
Endotheliosis, 674 
Enteritis, catarrhal, 116 

malignant, 118 

membranous, 112 

mucous, 112 
Enterokinase, 102 
Enteroliths, 118 
Enthelmintha, 139 
Entozoa in feces, 139 
Enumeration of blood cells, 455 , 465 , 468 

of pus cells, 354 
Eosin-hematoxylin stain, 479 

methylene-blue stain, 478 
Eosinophiles, 506 
Eosinophilia, 519 
Eosinophilic bronchitis, 10 
Epicritic elimination of nitrogen, 216 

polyuria, 179 
Epidemic cerebrospinal meningitis, 682 
Epiguanin, 240 
Episarkin, 240 
Epistaxis, Gull's renal, 355 
Epithelial casts, 359 

cells in feces, 119 

in gastric contents, 62 
in semen, 377 
in sputum, 11 
in urine, 349 
Erepsin, 102 

Error in cell counting, 466 
Erythrasma, 169 
Erythroblasts, 490 
Erythrocytes, 486 

appearance of, 486 

color-index of, 426 

counting of, 455 

crenation of, 487 

degenerations of, 498 

formation of, 386 

functions of, 501 

isotonicity of, 499 

nucleation of, 490 

number of, 492 

pathological types of, 488 

recognition of, in stains, 654 

resistance of, 499 

rouleaux formation of, 486 

shape of, 488 

size of, 488 

staining properties of, 496 

structure of, 486 

variations of, 492 
Erythrocytometer, 456 
Erythrocytosis, 495 
Erythrodextrin, 36, 82 
Esbach's method for albumin, 272 
Essential albuminuria, 261 
" pentosuria, 308 

renal hematuria, 355 
Esterification method of Fischer, 247 
Estivo-autumnal malaria, 566, 569 
Ethereal sulphates, 207, 210 
Euchlorhydria, 70 
Euglobulin, 275 
European cat-fluke, 159 



European hook-worm, 151 
Eustrongylus gigas, 160 

visceralis, 160 
Ewald test-meal, 54 
Ewald and Siever's method for gastric 

motility, 85 
Exercise, effect of, on red cells, 494 

leucocytosis due to, 517 
Extraction method for fat, 694 
Extraneous material in sputum, 9 
Extruded intracellulars, 564 
Exudates, 662 

bacteriology of, 667 

chyloid, 665 

chylous, 665 

conjunctival, 47 

cytology of, 672 

formation of, 662 

hemorrhagic, 665 

obtaining of, 663 

peritoneal, 675 

pleural, 674 

properties of, 663 

purulent, 666 

putrid, 666 

serofibrinous, 664 

serous, 664 

urethral, 668 

False albuminuria, 261 
Famine fever, 574 
Fasciola hepatica, 157 

humana, 157 
Fasciolopsis buski, 158 
Fasting stomach, contents of, 60 
Fat in blood, 449 

in exudates, 665 

in feces, 115, 123 

in milk, 693 

in urine, 346 
Fatty acids in blood, 449 

in exudates, 665 

in feces, 115, 123 

in sputum, 13 

in urine, 249 

casts, 359 

granules in leucocytes, 509 

stools, 107 
Favus, 165 
Febrile albuminuria, 264 

albumosuria, 280 

anemia, 538 

diseases, blood in, 549 

urine, 179 
Fecal vomitus, 61 
Feces, 99 

amount of, 104 

bacteriology of, 128 

bile acids in, 118 

biliary pigments in, 108, 117 

blood in, 108 

carbohydrates in, 116, 123 

chemical examination of, 121 

color of, 116 

concretions in, 118 

consistency of, 105 

crystals in, 120 

fat in, 114, 123 

food remnants in, 113 



INDEX. 



739 



Feces, formed, 105 

macroscopic examination of, 103 

marking of, 101 

microscopic examination of, 119 

morphological elements in, 119 

mucus in, in 

normal, 99 

odor of, 106 

parasitology of, 133 

protein in, 114 

pus in, 113 

reaction of, 121 

tissue fragments, in, 118 

total nitrogen of, 122 

total solids of, 121 

unformed, 105 
Fehling's test for glucose, qualitative, 
291 

quantitative, 295 
Female secretions, 380 
Fermentation method of Schmidt, 124 

test for diphtheria bacillus, 40 
for glucose, 293, 303 
Fermentative dyspepsia, 117 
Ferments in blood, 454 

in feces, 102 

in gastric juice, 64, 77 

in leucocytes, 454 

in sputum, 6 

intestinal, 102 

in urine, 251 

pancreatic, 102 
Ferrocyanide test for albumin, 271 
Ferrometer of Jolles, 453 
Fibers, elastic, 12, 115 

muscle, in feces, 115 
in gastric contents, 59 
Fibrin ferment, 402 

in blood, 404 

in urine, 283 

network, 404 

significance of, 404 

tests for, 283 
Fibrinogen, 402 
Fibrinous casts in sputum, 8 

in urine, 359 
Fibrinuria, 283 
Filaria Bancrofti, 577 

in blood, 577 

in urine, 346, 366 

nocturna, 577 

sanguinis hominis, 577 
Filariasis, 577 

Finely granular cells of Schultze, 504 
Fischer's esterification method, 247 

test-meal, 55 
Fish tape-worm, 144 
Fittipaldi's method for albumose, 279 
Fixation of complement, 617 

of smears, by chemicals, 475 
by heat, 473 
Fixed alkalinity, 187 
Flagella, staining of, 708 
Flagellata in feces, 137 

in sputum, 28 

in urine, 366 
Flat worms, 139 

Fleischl-Miescher hemometer, 417 
Flexner's serum, 683 



Florence's test for seminal fluid, 379 
Fluids, diluent for blood, 459, 466 
Fluke-worms, 145, 156 
Folin's method for acetone, 323 

for acidity of urine, 185 

for ammonia, 230 

for creatin, 245 

for creatinin, 243 

for free mineral acidity, 185 

for indican, 260 

for sulphates, 210 

for urea, 223 

for uric acid, 235 

standard diet, 101 
Folin and Denis' method for ammonia, 

444 

for non-protein nitrogen, 430 
urea, 435 
uric acid, 439 
Folin and Farmer's method for total 

nitrogen, 431 
Folin and Macallum's method for am- 
monia, 230 
Foreign bodies in sputum, 9 
Form of stools, 105 
Formaldehyd as preservative, 177, 696 
Formalin method for ammonia, 231 
Formation of blood, 386 

of casts, 356 

of exudates, 662 
Fraenkel's diplococcus, 25, 30, 589, 717 
Fragments of tissue in feces, 118 

in gastric contents, 63 

in sputum, 12 

in urine, 363 
Free hydrochloric acid, 65 

amount of, 69 

detection of, 66 

determination of, 67 

formation of, 51 

significance of, 70 

variations of, 70 
Freezing point of blood, 405 

of urine, 371 
Fresh blood, 470 
Friedlander's bacillus, 25, 46 
Frommer's test for acetone, 321 
Fucose, 308 
Functional albuminuria, 261 

diagnosis, 370 

hematuria, 355 
Functions of gastric ferments, 84 

of intestinal ferments, 102 

of leucocytes, 522 

of red cells, 501 
Fusaria mystax, 146 

vermicularis, 147 
Fusiform bacillus of Vincent, 40 
Futcher and Lazear's fixation method, 
476 

malarial stain, 484 

Gabbet's staining method, 21 
Gabritschewsky's polychromatophilia, 

497 

Gaffky's table, 23 
Galactosuria, 305, 312 
Gall stones in feces, 117 
appearance of, 117 



740 



INDEX. 



Gall stones, composition of, 117 
Gamete, 564 
Gametocyte, 564 
Gametoschizonts, 572 
Gas bacillus, 133 
Gases in blood, 454 
in feces, 125 
in gastric contents, 83 
Gastric carcinoma, 91 
contents, 50 

acetone in, 84 

after test meals, 62 

amino acids in, 83 

bacteria in, 62 

blood in, 59, 61, 83 

crystals in, 63 

digestion products in, 82 

epithelial cells in, 62 

food remnants in, 62 

from fasting stomach, 60 

from vomitus, 60 

gases in, 83 

indirect examination of, 87 

macroscopic examination of, 57 

microscopic examination of, 62 

mucus in, 60, 62, 91 

obtaining of, 52 

protozoa in, 63 

pus in, 62 

tissue fragments in, 63 
crises, 89 
juice, 57 

acetic acid in, 64, 73, 77 

acidity of, 64 

amount of, 57 

butyric acid in, 64, 73, 77 

combined hydrochloric acid in, 

71 
composition of, 64 

deficit of hydrochloric acid in, 73 

ferments of, 64, 78 

free hydrochloric acid in, 65, 92 

hyperacidity of, 65, 71, 88 

hypersecretion of, 89 

hypoacidity of, 65, 70 

in disease, 88 

lactic acid in, 74, 92 

organic acids in, 73 

Pawlow's work on, 51 

properties of, 57 

secretion of, 5 1 
motility, 84 
ulcer, 91 
Gastrin, 51 
Gastritis acute, 90 
atrophic, 90 
chronic, 90 
Gastrosuccorrhea, 89 
Gelatin, nutrient, 702 
Genital organs, secretions of, 376 
Genito-urinary tuberculosis, 352, 365 
Gerhardt's test for diacetic acid, 324 
Ghoreyeb's stain for spirochete, 670 
Giemsa's stain, 483, 670 
Gigantoblasts, 491 
Gigantocytes, 489 
Glanders, bacillus of, 27 
Globular decolorization, 497 
Globulin- albumin ratio, 275 



Globulin-albumin in blood, 275, 429 
in exudates, 663 
in milk, 693 
in urine, 274 

significance of, 275 
tests for, 275 
Glomerular insufficiency, 371 
Glossina palpalis, 575 
Glucose agar, 703 

in the blood, 446 
in the urine, 284 

determination of, 295 
significance of, 284 
tests for, 288 
Glutoid capsules, 103 
Glycemia, 446 
Glycerin agar, 703 
Glycocholic acid, 117, 191 
Glycocoll, 246 
Glycogen in the blood, 449. 
Glycosuria, 284 

alimentary, 285 
after poisoning, 287 
after use of drugs, 287 
diabetic, 286 
e saccharo, 284 
ex amylo, 284 
masked, 293 

neuro-hepatogenous, 286 
occult, 293 
physiologic, 285 
salt, 284 
transitory, 285 
Glycosuric acid, 333 
Glycuronic acid, 314 
Glycyl-tryptophan test, 93 
Gmelin's reaction for biliary pigments, 

330 
Goldhorn's stain, 580 
Goldschmiedt's test for glycuronic 

acid, 316 
Gonococcus, 667, 720 
Gonorrhea, complement-fixation test 

in, 637 
Gonorrheal conjunctivitis, 48 
stomatitis, 41 
threads, 353, 669 
urethritis, 669 
Goodman and Stern's method for albu- 
min, 273 
Gout, blood in, 548 

perinuclear granules in, 505 
urine in, 234 
Gowers' hemoglobinometer, 421 
Gram-negative organisms, 668 
Gram-positive organisms, 668 
Gram's etain, 668 
Granular casts, 357 

cells in blood, 504, 506, 507 
in prostatic fluid, 378 
in sputum, 10, n 
degeneration, 497 
Granules in blood, acidophile, 506 
basophile, 498, 506 
of Grawitz, 498 
Ehrlich's a, 506 
/?, 506 

r, 507 

8, 507 



INDEX. 



741 



Granules in blood, Ehrlich's s, 504 

eosinophile, 506 

fatty, 509 

glycogen, 509 

Grawitz, 498 

hemoconien, 525 

in malaria, 563, 565, 567 

mast cell, 506 

melanin, 563, 565, 567 

Neusser's, 505 

neutrophile, 504 

oxyphilic, 506 

perinuclear, 505 

sudanophile, 509 
in sputum, 11 
Grape-sugar in urine, 284 
Gravel in urine, 367 
Grawitz' basophilia, 498 
Green sputum, 3 
vomitus, 61 
Griess-Ilosvay reagent, 36 
Grinders' rot, 4 
Gross' method for trypsin, 102 
Ground itch, 152 
Gruber-Widal reaction, 607 
Guaiac test for blood, 109, no, 655 
Guanin, 240 

Gull's renal-epistaxis, 355 
Gum, animal in urine, 314 
Gummatous lymphoma, 545 
Gunning's mixture, 217 

test for acetone, 321 
Giinzburg's package, 87 
reagent, 66 

test for free hydrochloric acid, 66 
Gynecophorus haematobius, 367, 585 

Haeser's coefficient, 189 

Haines' coefficient, 189 

test for glucose, qualitative, 291 
quantitative, 299 

Haldane and Smith's method for 
volume of blood, 388 

Halitus sanguinis, 394 

Hammarsten's test for biliary pig- 
ments, 331 

Hammerschlag's method for specific 
gravity, 400 
for pepsin, 79 

Hap tines, 595 

Haptophore, 594 

Hard chancre, organism of, 669 

Hart's method for /?-oxybutyric acid, 
326 

Harvest bug, 162 

Hayem's solution, 459 

Hay fever, 45 

Hay's test for bile acids, 331 

Head louse, 163 

Heart disease cells, 1, 32 
pleurisy of, 675 

Heat fixation of smears, 473 

Heat test for albumin, 267 

Hehner-Maly method for organic acids, 

73 
Heller's table for examination of 
calculi, 369 
test for albumin, 268 
for hemoglobin, 282 



Hemameba malariae, 565 

vivax, 563 
Hemamebiasis, 561 
Hematemesis, 3, 61 
Hematin, 413 

hydrochlorate, 414, 658 
Hematoblasts, 523 
Hematochyluria, 578 
Hematocrit, 390 
Hematogenous albuminuria, 265 

albumosuria, 280 

urobilinuria, 255 
Hematoglobulin, 411 
Hematoidin in the blood, 414 

in sputum, n, 13 

in urine, 328 
Hematopoietic organs, 386, 525 
Hematoporphyrin in blood, 414 

in feces, 108 

in stains, 658 

in urine, 328 
Hematoporphyrinuria, 328 
Hematuria, 354 

angioneurotic, 355 

constitutional, 355 

essential, 355 

extra-renal, 355 

functional, 355 

idiopathic, 355 

renal, 355 • 
Hemin, 414, 658 
Hemoalkalimeter of Dare, 396 
Hemochromogen, 412 
Hemoconien, 525 
Hemocytometer of Durham, 467 

of Oliver, 468 

of Thoma-Zeiss, 456 
Hemoglobin in blood, 409 
amount of, 425 
derivatives of, 413 
estimation of, 415 
properties of, 409 
variations of, 425 

in sputum, 3, 5, n, 13 

in urine, 281, 328 
tests for, 282 

quotient, 426 

value, 426 
Hemoglobinemia, 281 
Hemoglobinemic degeneration, 499 
Hemoglobinometer of Dare, 420 

of Oliver, 422 

of Tallqvist, 424 
Hemoglobinuria, 281, 328 

paroxysmal, 281 

significance of, 281 

tests for, 282 
Hemolysins, 596 
Hemolysis, 596 
Hemolytic anemia, 537 
Hemometer of Fleischl-Miescher, 417 

of Sahli, 421 
Hemophilia, renal, 355 
Hemoptysis, 3 
Hemo-renal index, 452 
Hemorrhage, anemia due to, 535 

occult, 108 
Hemorrhagic exudates, 665 

nephritis, 355 



742 



INDEX. 



Hemosiderin, n, 415 

Hepatic insufficiency, 125, 222, 286, 305 

Hepatogenous albumosuria, 280 

urobilinuria, 255 
Herman-Perutz reaction, 651 
Herpes tonsurans, 166 
Heteroalbumosuria, 277 
Heterochylia, 89 
Heteroxanthin, 240 
Hexamitus duodenalis, 138 
Hexone bases, 83, 91, 247 
Hippuric acid in urine, 248, 345 
Histidin, 247 

Histoplasma capsulatum, 576 
Histoplasmosis, 576 
Hodgkin's disease, 544 
Hoffman's bacillus, 710 
Homogentisic acid, 333 
Hopkin's method for uric acid, 235 
Hoppe-Seyler's colorimetric pipet, 416 
Howell's immature nucleated reds, 490 

mature nucleated reds, 490 
Huppert-Messinger method for acetone, 

322 
Hyaline casts, 356 
Hydatid cysts, 143, 678 
Hydatidiform degeneration, 384 
Hydremia, 389 
Hydrobilirubin in stools, 107 
Hydrocele agar, 704 

fluid, 677 
Hydrochloric acid in gastric juice, 51 

amount of, 69 

combined, 71 

deficit, 73 

estimation of, 67 

free, 65 

physiologically active, 72 

tests for, 66 
Hydrogen sulphide in gastric contents, 
83. 

m urine, 208 
Hydronephrosis, 678 
Hydrops folliculorum Graafii, 676 
Hydroquinone-acetic acid, 333 
Hydruria, 178 
Hymenolepis diminuta, 142 

flavopunctata, 142 

murina, 142 

nana, 142 
Hypalbuminosis, 428 
Hyperacidity of gastric juice, 65, 71 
Hyperalbuminosis, 428 
Hyperchlorhydria, 71, 88 
Hyperglycemia, 446 
Hyperinosis, 404 
Hypermotility of stomach, 85 
Hypersecretion of gastric juice, 89 
Hypertonic solutions, 500 
Hyphogenous sycosis, 166 
Hypinosis, 404 
Hypochlorhydria, 70 
Hypostatic albuminuria, 264 
Hypotonic solutions, 500 
Hypoxanthin, 240 

Idiopathic enterogenous cyanosis, 412 

pentosuria, 308 
Ilosvay's reagent, 36 



Immature nucleated reds of Howell, 

490 
Immunity, 593 
Inactivation of serum, 596 
Inanition, anemia due to, 536 
Incubation, 704 
Index, bacillary, 599 

color, 426 

hemoglobin, 426 

opsonic, 599 

phagocytic, 599 

volume, 391 
India ink method for spirochete, 670 
Indican, 256 
Indicanuria, 256 
Indigo blue in urine, 256 

red in urine, 258 
Indirect examination of gastric con- 
tents, 87 
Indol-acetic acid, 260 
Indoxyl-potassium sulphate in urine, 

, • 256 
Infectious diseases, blood in, 549 

Influenza, bacillus of, 25, 711 

sputum in, 33 
Infusoria, in feces, 138 

in sputum, 28 

in urine, 366 
Inorganic constituents of blood, 452 

of urine, 192 
Inoscopy, 667 
Inosite in urine, 314 
Insecta, 163 
Insufficiency, digestive, 113 

glomerular, 371 

hepatic, 125, 222, 286, 305 

motor, 84 

renal, 371 

tubular, 371 
Intermittent albuminuria, 263 
Intestinal concretions, 118 

digestion, 102 

juices, 102 

obstruction, 114 

parasites, 133 

sand, 118 
Iodide of potassium test, 86 
Iodoform test for acetone, 321 

for lactic acid, 76 
Iodophilia, 509 
Iron in the blood, 453 

in urine, 213 
Irritation forms of leucocytes, 509 
Isoagglutinins, 653 
Isohemolysins, 653 
Isomaltose in urine, 314 
Isotonicity of red cells, 499 
Isotonic solutions, 499 
Itch parasite, 162 

Jaeger and Heubner's diplococcus, 683 
Jaffe's test for creatinin, 243 

for indican, 257 
von Jaksch's anemia, 533 
Japanese liver-fluke, 159 
Jaundice, blood in, 452 

sputum in, 4 

urine in, 182, 329 
Jecorin, 445 



INDEX. 



743 



Jenner's stain, 481 
Jigger, 165 

Jolles' ferrometer, 453 
Jousset's fluid, 667 
Juice, gastric, 57 

intestinal, 101 
Justus' test for syphilitic blood, 559 

Kahler's disease, 277 
Kala-azar, blood in, 576 

parasite of, 576 
Karyomorphism of neutrophiles, 505 
Kastle and Loevenhart's method for 

lipase, 252 
Kathrein's test for bile pigments, 330 
Kelling's test for lactic acid, 75 
Kendall and Day's method for typhoid 

bacilli, 131 
Kidney, abscess of, 352 

acute inflammation of, urine in, 
178, 180, 190, 194, 201, 217, 
266 
amyloid disease of, urine in, 178, 

181, 253, 266, 275 
cancer of, 354, 363 
chronic inflammation of, urine in, 
178, 179, 181, 190, 194, 201, 
266 
echinococcus cysts of, 366 
hemorrhagic lesions of, 354 
hydronephrotic cysts of, 678 
malignant disease of, 354, 363 
stones, 367 

suppurative lesions of, 352 
syphilitic disease of, 266 
tubercular, 352 
Kjeldahl's method for nitrogen, 217 
Klebs-Loffler bacillus, 38, 709 
Knop-Hlifner method for urea, 222 
Koch's bacillus, 18, 132, 364, 592, 667 
comma bacillus, 61, 129, 715 
tuberculin, 18, 601 
Koch-Weeks bacillus, 48 
Kohlrausch's method for electric con- 
ductivity, 371, 406 
Kowarsky's method for sugar in blood, 

447 

Krabbea grandis, 145 
Kreatin (see Creatin), 241 
Kreatinin (see Creatinin), 241 

Lab, 64, 81 

Labor, albuminuria following, 262 
Lactic acid in blood, 450 
in gastric contents, 74 

in carcinoma, 92 

significance of, 74 

tests for, 75 
in urine, 249 
Lactose in milk, 695 

in urine, 312 
Lactosuria, 312 
Lactose-litmus agar, 703 
Laiose, 306 
Laking of blood, 394 
Lamblia intestinalis, 138 
Landau's test, 652 
Lange's colloidal-gold test, 686 



Large lymphocytes, 502 

mononuclear leucocytes, 503 
Larvae in aural secretions, 47 

in feces, 148, 149, 153 
Laveran's malarial organism, 561 
Layers of sputum, 5 
Lead, anemia due to, 538 

basophilia in poisoning, 498 
Lecithin globules in semen, 377 
Legal's test for acetone, 320 
Leiner's test for casein, 115 
Leishman-Donovan bodies, 576 
Le Nobel's test for acetone, 320 
Leo's method for chymosin, 81 
Leprosy, bacillus of, in sputum, 24 

blood in, 559 
Leptodera intestinalis et stercoralis, 

147 
Leptothrix buccalis, 38 

in sputum, 15 
Leptus autumnalis, 162 
Leube's test of gastric motility, 85 
Leucin in sputum, 13 

in urine, 246, 343 
Leucocytes, 502 

appearance of, 502 

basophilic, 506 

counting of, 465 

degenerated forms of, 509 

differential counting of , 510 

eosinophilic, 506 

ferments of, 454 

formation of, 387 

functions of, 522 

granules in, 498, 506, 507 

in blood, 502 

in exudates, 673 

in feces, 120 

in gastric contents, 62 

in milk, 698 

in sputum, 10 

in urine, 351 

irritation forms, 509 

karymorphism of, 505 

large mononuclear, 503 

lymphocytes, 502 

mast-cell, 506 

myelocytes, 507 

neutrophilic, 504 

number of, 511 

oxyphilic, 506 

pigmented, 568 

polymorphonuclear, 504 

small mononuclear, 503 

splenocytes, 503 

transition forms of, 504 

types of, 502 

variations in number of, 511, 521 
Leucocytic crystals in sputum, 14 
Leucocytometer, 457 
Leucocytosis, 512 

agonal, 516 

antemortem, 516 

cachectic, 515 

eosinophilic, 5 [9 

infectious, 515 

inflammatory, 515 

mast-cell, 521 

mixed, 518 



744 



INDEX. 



Leucocytosis of digestion, 513 

of pregnancy, 514 

of the new-born, 514 

polymorphonuclear, 512 

post-hemorrhagic, 516 

therapeutic, 516 
Leucohydrobilirubin in feces, 107 
Leucopenia, 521 
Leucorrhea, 381 
Leucourobilin, 107 
Leukanemia, 534 
Leukemia, 539 

acute, 543 

lymphatic, 543 

mixed, 539 

splenomyelogenous, 539 
Levaditi and Manouelian's method for 

staining treponema, 671 
Levulose in urine, 305 

determination of, 306 

recognition of, 306 

significance of, 305 

tests for, 306 
Levulosuria, 305 
Lieben's test for acetone, 321 
Lientery, 113 

Limitations of blood examinations, 659 
Limnaea truncatula, 158 
Lipacidemia, 450 
Lipaciduria, 249 
Lipase in gastric juice, 64, 82 

in pancreatic juice, 102 

in urine, 252 
Lipemia, 450, 547 
Lipliawsky's test for diacetic acid, 

T . .325 

Lipuria, 346 

Liquor sanguinis, 393 

Lithemic diathesis, 234 

Litmus-glucose agar, 703 

lactose agar, 703 

milk, 704 
Liver, abscess of, sputum in, 33 

insufficiency of, 125, 222, 286, 305 
Lobar pneumonia, blood in, 550 

chlorids in urine of, 194 

organism of, 25, 31 

sputum in, 31 
Lochia alba, 383 

cruenta, 383 

rubra, 383 

serosa, 383 
Loffler's antiformin method, 18 

methylene blue, 21 
Lohnstein's saccharometer, 303 
Long's coefficient, 189 
Lordotic albuminuria, 263 
Luetin reaction, 603 
Lumbar puncture, 679 
Lung, abscess of, sputum in, 33 

fluke in sputum, 29 

inflammation of, 31, 194, 550 

stones, 8 
Lymphatic leukemia, 543 

pseudoleukemia, 544 
Lymphemia, 543 
Lymphocytes, 502 
Lymphocytosis, 518 
Lymphopenia, 518 



Lymphosarcoma, 545 
Lysins, 596 
Lytic action, 596 

Macrocytes, 489 
Macrocythemia, 489 
Macrocytosis, 489, 531 
Macrogamete, 565 

Magnesium ammonium phosphate in 
feces, 120 

in sputum, 13 

in urine, 348 

phosphate in urine, 198, 348 

salts in urine, 211 

soaps in feces, 116 
Malaria, blood in, 572 

fresh blood in, 563 

mosquito theory of, 561 

parasites of, 563 

estivo-autumnal, 566, 569 
quartan, 565, 569 
tertian, 563, 568 

stained smears in, 568 
Malarial pigment, 415 
Male secretions, 376 
Malfatti's method for ammonia, 231 
Malignant disease, blood in, 559 
gastric juice in, 91 
urine in, 354, 366 

lymphoma, 545 

pleurisy, 675 
Mallory's stain for Negri bodies, 173 
Malone's test for pregnancy, 247 
Maltose in urine, 313 
Mammary secretions, 688 
Maragliano's endoglobular degenera- 
tion, 497 
Marechalt's test for bile pigments, 330 
Marshall's method for urea, 225 
Martius and Liittke's method for HC1, 

7i 
Marx's fluid, 654 
Mast- cell granules, 506 

leucocytosis, 521 
Masturbators, albuminuria of, 263 
Mature nucleated reds of Howell, 490 
May-Griinwald stain, 482 
McCrudden's method for calcium, 212 
Meals, test, 54 
Measles, blood in, 554 
Medicinal leucocytosis, 516 
Medico-legal aspects of blood, 654 

of semen, 378 
Megaloblasts, 491 
Megalocytes, 489 
Megalogastria, 85 
Megastoma entericum, 138 

intestinale, 138 
Melanin, 182, 332, 415 
Melanogen, 182 
Mel anuria, 332 
• Membranous dysmenorrhea, 383 

enteritis, 112 

ureteritis, 252 
Meningeal fluid, examination of, 678 
Meningitis, epidemic cerebrospinal, 682 

tubercular, 684 
Meningococcus of Bonome, 683 

of Weichselbaum, 683, 721 



INDEX. 



745 



Menstruation, 382 

Messinger method for acetone, 322 

Metalbumin in ovarian cysts, 677 

Metamyelocytes, 505 

Methemoglobin, 411 

Methylene azure stains, 481 

blue in urine, 183, 335 
stains, 21, 478 

test for functional activity, 372 
Methylphenylosazon, 307 
Methylxanthin, 240 
Mett's method for pepsin, 79 
Meyer's test for blood, 656 
Microblasts, 490 

Micrococcus, catarrhalis in sputum, 17, 
722 

tetragenus in sputum, 17 
Microcytes, 488 
Microgametes, 565 
Microgametocytes, 565 
Microscopy of blood, 469 

of exudates, 672 

of feces, 119 

of gastric contents, 62 

of milk, 696 

of semen, 376 

of sputum, 9 

of urine, 336 
Microsporon Audouini, 167 

furfur, 167 

minutissimum, 169 

scorteum, 166 
Miescher's hemoglobinometer, 417 
Milk, 688 

appearance of, 690 

ash of, 691 

bacteriology of, 696 

coagulation of, 691 

composition of, 689 

cow's, 689 

curds in stools, 115 

fat of, 693 

human, 689 

lactose of, 695 

microscopy of, 696 

preservatives in, 695 

properties of, 689 

protein of, 691 

reaction of, 691 

specific gravity of, 690 

sugar of, 695 

total solids, 691 
Milk-curdling ferment, 64, 81 
Milky zone, 563 
Mineral acidity of urine, 185 
Mintz's method for free HC1, 68 
Mixed infection in tuberculosis, 23 

leucocytosis, 518 
Molds in aural secretions, 47 

in buccal secretions, 36 

in sputum, 16 
Moller's method for spores, 708 
Monocalcium phosphate in urine, 346 
Monocercomonas hominis, 138 
Monochromatophilia, 496 
Mononuclears, basophile, 508 

eosinophile, 508 

large, 502 

neutrophile, 508 



Mononuclears, small, 502 

Moore and Wilson's test for alkalinity, 

395 

Morax-Axenfeld diplobacillus, 48 
Morner-Sjoqvist method for urea, 225 
Morner's mucin-like bodies in urine, 253 

test for tyrosin, 345 
Morning sputum, 1 
Moro's tuberculin reaction, 601 
Morphology of blood, 469 

of blood-forming organs, 525 
Mosquito, anopheles, 561 

culex, 583 

cycle of malarial parasites, 571 

stegomyia, 583 

theory of malaria, 561 
of yellow fever, 583 
Motility of intestine, 104 

of stomach, 84 
detection of, 85 
types of, 84 
Motor insufficiency, 84 
Moults, 161 

Mouth, inflammation of, 40 
catarrhal, 40 
gonorrheal, 41 
mycotic, 41 
ulcerative, 40 
ulceromembranous, 40 

secretions of, 35 
Much's method for tubercle bacilli, 22 
Mucin in ovarian cysts, 677 

in sputum, 5 

in urine, 252 
Mucinophiles, 507 
Mucoid material in urine, 349 

sputum, 5 
Mucopurulent sputum, 5 
Mucor in sputum, 16 
Mucous corpuscles, 349 

threads in urine, 353, 669 
Mucus in feces, in 

appearance of, in 
detection of, 1 1 1 
significance of, 112 

in gastric contents, 60, 62, 91 

in sputum, 5 

in urine, 252 
Miillern's blood stain, 478 
Murexid test, 339 
Muscle fibers in feces, 114 

in gastric contents, 60 

in sputum, 12 
Mycelial casts in sputum, 8 
Myelemia, 539 

Myelin granules in sputum, 11 
Myeloblasts, 503 
Myelocytes, 507 

basophile, 508 

eosinophile, 508 

neutrophile, 508 
Cornil's, 508 
Ehrlich's, 508 
Myelocytosis, 518 
Myelogenous leukemia, 539 
Myeloid leukemia, 539 
Myelopathic albumosuria, 277 
Myxedema, blood in, 549 
Myxococcidium stegomyiae, 583 



746 



INDEX. 



Myxoid cyst of ovary, 676 
Myxoma of the placenta, 384 

Nakayama's test for bile pigments, 330 
Naphthoresorcin test, 316 
Nasal secretion, 44 

bacteria in, 44 

composition of, 44 

concretions in, 46 

pathology of, 44 

spinal fluid in, 45 
Negri bodies, 172 
Neisser's diplococcus, 667, 720 

stain for diphtheria bacillus, 39 
Nematodes in feces, 145 

in urine, 366 
Nephritic albuminuria, 265 

hematuria, 355 

oliguria, 179 

pleuris3^, 675 
Nephritis, acute, 178, 180, 190, 194, 
201, 217, 265 

albuminuria of, 265 

chronic diffuse, 190, 194, 201, 265 
interstitial, 178, 182, 265 
parenchymatous, 178, 180, 265 

hemorrhagic, 355 

suppurative, 352 

syphilitic, 266 

unilateral, 371 
Nervous dyspepsia, 90 

type of albuminuria, 265 
of polyuria, 179 
Neubauer and Fischer's test for gastric 

carcinoma, 93 
Neuberg and Wohlgemuth's method for 

pentose, 310 
Neuberg' s test for glycuronic acid, 316 

for levulose, 306 
Neusser's granules, 505 
Neutral calcium phosphate in urine, 
346 

dyes, 477 

stains, 477 

sulphur in urine, 208 
Neutrophile cells, 504, 508 

granules, 504 
Neutrophilic karyolobism, 505 
New-born, albuminuria of, 262 

leucocytosis of, 514 
Night urine, 178 
Nikiforoff's method of fixation of 

smears, 475 
Nitric acid test for albumin, 268 
Nitrites in saliva, 36 
Nitrogen of sputum, 2 

of urine, 213 

partition of blood, 430 
of urine, 214 
Nitrogenous balance, 213 

bodies in blood, 430 

in exudates, 664 

in feces, 122 

in gastric contents, 93 

in milk, 692 

in sputum, 2, 5 

in transudates, 664 

in urine, 213 
allantoin, 248 



Nitrogenous bodies in urine, alloxypro- 
teic acid, 208, 248 
amino-acids, 246 
ammonia, 227 
creatinin, 241 
hippuric acid, 248, 345 
oxyproteic acid, 248 
purin bases, 240 
total, 213 
undetermined, 246 
urea, 219 
uric acid, 232 
equilibrium, 213 
Nitroprussid test for acetone, 320 
Nocht's malarial stain, 484 
Noguchi's antigen, 622 
butyric acid test, 685 
luetin reaction, 603 
method for spirochete, 581, 671 
modification of Wassermann test, 
629 
Nondiffusible alkalinity of blood, 395 
Nonne's test of cerebrospinal fluid, 

685 
Nonprotein nitrogen of blood, 430 
Normal feces, 99 

salt solution, 500 
Normoblasts, 490 
Normocytes, 486 
Nose, secretions of, 44 
Nubecula, 180 
Xubecular threads, 180, 349 
Nucleated red cells, 490 

Howell's immature, 490 
mature, 490 
Nuclein bases, 122, 191, 240, 341 
Nucleinic acid, 232, 253 
Xucleo-albumin in urine, 252 
Number of blood plates, 524 
of leucocytes, 511 
of red cells, 492 
of stools, 104 

of tubercle bacilli in sputum, 22 
Nummular sputum, 5, 6 
Nutrient agar, 702 
broth, 701 
gelatin, 702 
Nutrition, effect of, on blood, 493, 

536 
Nycturia, 178 
Nylander's test for glucose, 292 

Obermayer's test for indican, 257 
Obermeier's spirillum, 574 
Obtaining blood, 392, 585 

exudates, 663 

gastric contents, 52 
Occult blood in feces, 108 
Ochronosis, 333 
Odor of blood, 394 

of exudates, 664 

of feces, 106 

of gastric contents, 59 

of sputum, 4 

of urine, 183 
Oidium albicans, 16, 41 
Oil test-breakfast, 102 
Oligemia, 388 
Oligochromemia, 426 



INDEX. 



747 



Oligocythemia, 495 

Oliguria, 179 

Oliver's hemocytometer, 468 

hemoglobinometer, 422 

test for bile acids, 332 
Oocyst, 572 
Ookinet, 570 

Operation, blood after, 546 
Opisthorchis felineus, 159 

sinensis, 159 
Oppler-Boas bacillus, 62 
Opsonic index, 599 
Opsonins, 598 
Optical activity of carbohydrates, 300 

of conjugated glycuronates, 301 

of glucuronic acid, 301 

of urine, 190 
Oral secretions, 35 
Orcein stain, 12 
Orcin test for pentose, 310 
Organic acidity of urine, 185 

acids in gastric contents, 73 
in urine, 249 
Organized sediments in urine, 349 

bacteria, 363 

blood cells, 354 

casts, 356 

epithelial cells, 349 

mucoid material, 349 

parasites, 366 

pus cells, 351 

spermatozoa, 363 

tissue fragments, 363 
Origin of casts, 356 

of leucocytes, 387 

of red cells, 386 
Orthostatic albuminuria, 263 
Orthotic albuminuria, 263 
Osier's disease, 496 
Osmotic pressure of blood, 405 

of urine, 371 
Otomycosis, 47 
Ova in feces, 133, 154 

in sputum, 29 

in urine, 367 

of anopheles, 561 

of intestinal parasites, - 154 
Ovarian cysts, 676 

colloid, 676 

dermoid, 677 

myxoid, 676 

papillary, 677 

serous, 676 
Ovoids in malarial blood, 567 
Oxalate of calcium in sputum, 14 

in urine, 250, 341 

calculi, 368 
Oxalic acid in urine, 250 

amount of, 250 

determination of, 251 

origin of, 250 

variations of, 250 
Oxaluria, 250 
Oxaluric acid, 250 
Oxid of bismuth in feces, 107 
/3-oxybutyric acid in urine, 325 

determination of, 326 

significance of, 326 

tests for, 326 



Oxyhemoglobin, 411 

p-oxyphenyl- a- amino-propionic acid, 

344 
Oxyphilic cells, 496 

granules, 506 
Oxyproteic acid, 191, 248 
Oxyuris vermicularis, 147 
Ozena, 46 

Palpation, albuminuria due to, 264 
Paludism, 561 
Pancreatic cysts, 678 

disease, feces in, 114 
fluid, 678 
juice, 102 

composition of, 102 
ferments of, 102 
insufficiency of, 114 
Panoptic staining, 478, 481 
Papillary cysts of the ovary, 677 
Pappenheim's amblyochromatic ery- 
throblasts, 491 
heteroplastic promyelocytes, 508 
method for tubercle bacillus, 21 
stain for blood smears, 481, 484 
trachyochromatic erythroblasts, 

, 490 
Paracresol, 333 

Paragonimus westermanii, 29 

Paramecium coli, 138 

Paramucin, 677 

Parasites, anemia due to, 537 

eosinophilia due to, 520 

in blood, 561 

in feces, 133 

in sputum, 28 

intestinal, 133 

in urine, 366 

malarial, 561 

of the skin, 161 
Parasitology of the blood, 561 

of the feces, 133 

of the skin, 161 
Paratyphoid bacillus, 131, 588 
Paraxanthin, 240 

Parenchymatous nephritis, acute, 178, 
180, 190, 194, 201, 217, 266 

chronic, 178, 179, 266 
Parhemoglobin, 409 
Parovarian cysts, 677 
Paroxysmal hemoglobinuria, 281 

polyuria, 179 
Pathogenic bacteria in blood, 585 

in exudates, 667 

in feces, 128 

in gastric contents, 62 

in milk, 696 

in sputum, 17 

in urine, 363 
Pea-soup stools, 130 
Pedi cuius capitis, 163 

pubis, 163 

vestimenti, 163 
Penicillium glaucum, 16 
Pentose in urine, 308 

determination of, 310 

significance of, 308 

tests for, 309 
Pentosuria, alimentary, 308 



74 8 



INDEX. 






Pentosuria, essential ,^308 J 

idiopathic, 308 

intrinsic, 308 
Penzoldt and Faber's test' 86 
Pepsin in gastric juice, 64, 77 
activity of, 77 
detection of, 78 
determination of, 79 
significance of, 78 

in urine, 251 
Pepsinogen, 77 
Peptic glands, 50 
Peptone in the blood, 429 

in gastric contents, 82 

in urine, 281 
Peptonuria, 281 
Perez' bacillus, 46 

Perforating empyema, sputum in, 34 
Pericardial fluid, 662 
Perinuclear granules of Neusser, 505 
Periodic albuminuria, 263 

polyuria, 179 
Peritoneal exudates, 675 

composition of, 675 

cytology of, 675 
Permeability of red cells, 405, 499 

renal, 370 
Pernicious anemia, 531 
Pertussis, blood in, 556 

organism of, 26, 711 
Pessary forms of red cells, 486 
Petreff 's method for tubercle bacilli, 19 
Pettenkofer's test, 332 
Pfeiffer's bacillus, 26, 33, 711 
Phagocytic cells, 4, 598 

index, 600 
Phagocytosis, 598 
Pharyngomycosis leptothrica, 38 
Phenol in feces, 99 

in urine, 332 
Phenolphthalin test, 656 
Phenolsulphuric acid, 332 
Phenolsulphonephthalein test, 373 
Phenoltetrachlorphthalein test, 125 
Phenylglucosazon, 294 
Phenylhydrazine test for glucose, 294 
Phloridzin test, 373 
Phloroglucin test for pentose, 309 

vanillin test for HC1, 66 
Phosphates, calcium, 199, 212 

in blood, 453 

in sputum, 13 

in urine, 198 

magnesium, 198, 213, 348 

magnesium-ammonium, 13,120,348 

triple, 13, 120, 348 
Phosphatic calculi, 368 

diabetes, 201 

sediments in urine, 346, 347 
Phosphaturia, 200, 347 
Phosphorus containing proteins, 253 

poisoning, blood in, 538 
urine in, 221 
Phthirius inguinalis, 163 
Phthisis, blood in, 557 

hemoptysis in, 3 

melanotica, 11 

sputum in, 30 

stone-cutters', 4 



Physiological albuminuria, 261 
glycosuria, 284 
salt solution, 500 

variations in blood cells, 492, 513 
Physiologically active HC1, 72 
Physis intestinalis, 153 
Pigment, bile, in blood, 451 

in feces, 108, 117 _^ 

in gastric contents, 59, 61 
in sputum, 3 
in urine, 329 
blood, in feces, 108 

in gastric contents, 59, 61/83 
in sputum, 3, 5, 11, 12, 14 
in urine, 328 
coal, in sputum, 4, 34 
in leucocytes, 4, 573 
in red cells, 497, 563 
of blood, 409 
of urine, 254, 328 
Pin worm, 147 
Pineapple test, 77 
Piroplasma hominis, 584 
Piroplasmosis, 584 

von Pirquet's tuberculin reaction, 602 
Placenta cells, 384 
Plague bacillus, 27 
Plasma, 393 

Plasmodium falciparum, 56(1 
quotidianum, 565 
malariae, 565 
precox, 566 
tenue, 563 
vivax, 563 
variety minuta, 563 
Platelets, blood, 523 
Platodes in feces, 139 
Plehn's karyochromatophilic granules, 

569 
Plethora, cellular, 389 

serous, 389 

true, 389 

vera, 389 
Pleuritic effusions, 674 

cytology of, 674 

withdrawal of, 663 
Plugs, Dittrich's, 6 

prostatic, 378 
Pneumococcus of Fraenkel, 25, 31, 589, 
717 

pleurisy, 674 
Pneumoliths, 8 
Pneumonia, blood in, 550 

chlorids in urine in, 194 

organism of, 25, 31 

sputum in, 31 

urine in, 194 
Pneumonoconioses, 34 
Pneumonomycosis aspergillina, 16 
Poikilocytes, 489 
Poikilocytosis, 489 
Poisons, blood, 538 
Polariscope, 300 

Polariscopic method for glucose, 302 
Poliomyelitis, acute anterior, 684 
Polychromasia, 496 
Polychromatophilia of Gabritschewsky, 

497 
of Maragliano, 497 



INDEX. 



749 



Polychrome dyes, 481 
Polychromemia, 495 
Polyglobulia, 495 
Polymorphonuclear basophiles, 506 

eosinophiles, 506 

neutrophiles, 504 
Polymorphonuclear neutrophiliosis, 512 
Polynucleosis, 512, 674 
Polyplasmia, 389 
Polyuria, 178 

epicriiic, 179 

paroxysmal, 179 

periodic, 179 
Poor, anemia of the, 536 
Pork tape-worm, 140 
Posterior urethritis, 353 
Post hemorrhagic anemia, 535 

infectious albuminuria, 264 
Postural albuminuria, 263 
Potassium acid urate sediment, 340 

ferrocyanide test for albumin, 271 

iodide test, 86 

of blood, 409 

of urine, 211 

sulphocyanate in saliva, 35 
Precipitinophore, 598 
Precipitins, 598 
Precipitin test for blood, 614 
Pregnancy, albuminuria of, 262 

ammonia in urine of, 228 

anemia of, 536 

blood in, 514 

leucocytosis of, 514 

sero-diagnosis of, 638 

urine test in, 247 
Preparation of blood smears, 471 

culture media, 701 

vaccines, 724 
Preservation of urine, 176 
Primary anemia, 528 

pernicious anemia, 531 

proteoses, 276 

tubercular pleurisy, 674 
Products of gastric digestion, 82 

. of intestinal digestion, 102 
Progressive pernicious anemia, 531 
Promyelocytes, 508 
Propepsin, 51, 78 
Prostatic casts, 378 

fluid, 376 

plugs, 378 

secretion, 376 
Prostatitis, 353 
Prostatorrhea, 378 
Protalbumose, 276 
Protein in blood, 427 

in exudates, 664 

in feces, 114 

in gastric contents, 82, 93 

in milk, 692 

in sputum, 5 

in urine, 260 

quotient of serum, 429 
Proteoses in urine, 276 
Prothrombase, 402 
Protoryxomyces coprinarius, 137 
Protozoa in blood, 561 

in feces, 134 

in gastric contents, 63 



Protozoa in sputum, 28 

in urine, 366 
Provocative Wassermann test, 636 
Prowazek-Greeff trachoma bodies, 48 
Prune-juice sputum, 3, 31 
Pseudo casts, 361 

diphtheria bacillus, 40, 710 

elastic tissue, 12 

gall stones, 118 

globulin, 275 

hemoglobin, 411 

leukemia, 544 
infantum, 533 

mucin, 677 

nucleation, 497 

parasites, 153 

rhabditis stercoralis, 147 
Ptomaines in feces, 123 

in urine, 342 
Ptyalin, 35, 36, 82 
Ptyalism, 37 

Puberty, albuminuria of, 264 
Puerperal infection, 382 
Pulex irritans, 165 

penetrans, 165 
Pulmonary actinomycosis, 27 

gangrene, 33 

hemorrhage, 3 

tuberculosis, 30 
Punctate basophilia of Grawitz, 498 
Purdy's method for albumin, 274 

for chlorids, 198 

for glucose, 298 

for phosphates, 205 

for sulphates, 210 
Purin bases in urine, 191, 240, 341 
Purpura hemorrhagica, blood in, 531 
Purpurin, 254 
Purulent exudates, 666 

sputum, 5 

urine, 351 
Pus casts, 359 

cells in feces, 113 

in gastric contents, 62 
in sputum, 10 
in urine, 351 

enumeration of, 354 
significance of, 353 
tests for, 353 
Putrescin, 123 
Putrid bronchitis, 32 

exudates, 666 
Pycnometer, 188, 400 
Pycnotic nucleus, 490 
Pyelitis, urine in, 351 

productiva, 252 
Pyelonephritis, 351 
Pyloric glands, 50 

stenosis, 86, 92 
Pyogenic albumosuria, 280 
Pyonephrosis, 351 
Pyorrhea alveolaris, 42 
Pyrocatechin, 333 
Pyuria, 351 



Quantity of blood, 387 
of gastric juice, 57 
of urine, 177 

Quartan malarial parasite, 



565 



75o 



INDEX. 



Quartan, asexual cycle of 565 

sexual cycle of, 566 
Quotient, albumin, 275, 428 

protein, 275, 429 

volume, 391 

Ratio of N to CI output, 193 

to P 2 6 output, 200 

to S0 3 output, 206 
Ray fungus, 27 
Reaction of blood, 395 

of feces, 121 

of gastric contents, 57 

of milk, 691 

of spinal fluid, 680 

of sputum, 3 

of urine, 184 
Reactivity of blood, 395 
Receptors, 595 

Rectum, blood in cancer of, 108 
Red cells (see Erythrocytes), 486 

in exudates, 665 

in feces, 109, 120 

in gastric contents, 59, 61, 83 

in sputum, 3, 5, 11 

in suspected stains, 654 

in urine, 354 
Red, indigo in urine, 258 

jigger, 162 

sputum, 3 
Refraction coefficient of serum, 389, 429 
Reichmann's disease, 89 
Relapsing fever, 574 
Relative value of phosphoric acid, 201 
Removal of albumin, 274 

of glucose, 294, 303 

of turbidity, 267 
Renal abscess, 351 

albumosuria, 280 

aneurism, 355 

calculus, 367 

concretions, 367 

diabetes, mellitus, 284, 373 

diagnosis, 370 

epistaxis, 355 

epithelial cells in urine, 349 

hematuria, 355 

hemophilia, 355 

insufficiency, 370 
Rennin, 64, 81 
Resistance of red cells, 499 
Resorcin test for free HC1, 67 
Rhabdonema intestinalis, 147 

strongyloides, 147 
Rhamnose in urine, 308 
Rheumatism, blood in, 557 
Rhinitis, 45 

Rhizopoda in feces, 134 
Rice-water stools, 106, 129 

vomitus, 61 
Rickets, blood in, 549 
Ricketts' organism of spotted fever, 584 
Riegel's method for chymosin, 81 

test meal, 55 
Riggs' disease, 42 
Ring bodies in red cells, 498 
of Cabot, 498 

worm of the beard, 166 
of the body, 166 



Ring worm of the scalp, 167 

Ringer's solution, 500 

Rivalta's test for exudates, 664 

Roberts' method for glucose, 304 

Roch's test for albumin, 271 

Rocky Mountain spotted fever, blood 

in, 584 
organism of, 584 
Romanowsky's stain, 481 
Ronchese's method for ammonia, 231 
Rosacic acid, 254 
Rosenbach's method for bile pigments, 

330 
for skatoxyl, 258 
Rosenow's capsule stain, 590 
Rosin's test for bile pigments, 330 
Ross-Jones test of cerebrospinal fluid, 

686 
Rot, grinders', 4 
Rouleaux formation, 486 
Round worms in feces, 145 
Rowntree and Geraghty's test, 373 
Rubner's test for lactose, 313 
Rudisch and Kleeberg's method for 

uric acid, 237 
Rudolf's method for coagulation time, 

403 

Ruhemann's uricometer, 239 
Russell and Brodie's coagulometer, 403 
Russo's test for typhoid, 335 
Rusty sputum, 3, 31 

Saccharometer of Einhorn, 303 

of Lohnstein, 303 
Saccharomyces cerevisiae, 15, 288 
Saccharose in urine, 314 
Sagitula hominis, 153 
Sago-like granules in sputum, I, II 
Sahli's desmoid reaction, 87 

hemometer, 421 

test-meal, 56 
Salicylic acid as preservative, 696 

test for gastric motility, 85 
Saliva, 35 

amount of, 35 

bacteria in, 36 

cells in, 36 

chemistry of, 35 

ferments in, 35 

microscopic examination of, 36 

nitrites in, 36 

obtaining of, 37 

pathologic changes in, 37 

potassium sulphocyanate in, 35 

ptyalin in, 35, 36 
Salivary corpuscles, 36 
Salivation, 37 
Salkowski method for alkalinity of 

blood, 397 
Salkowski-Ludwig method for uric 

acid, 236 
Salol test of Ewald and Sievers, 85 
Salomon's test for gastric carcinoma, 93 
Salt glycosuria, 284 
Salzer's test-meal, 56 
Sand flea, 165 

intestinal, 118 

in urine, 367 

renal, 367 



INDEX. 



7Si 



Sanguinous exudates, 665 

sputum, 5 
Saprophytes in feces, 128 

in sputum, 14 

in urine, 363 
Sarcinse in feces, 129 

in gastric contents, 63 

in sputum, 17 

in urine, 363 

ventriculi, 63 
Sarcoma, blood in, 545 
Sarcoptes scabiei, 162 
Saturation deficit, 73 
Scarlet fever, blood in, 553 
Schaer's test for blood, 109, 655 
Scherer's method for albumin, 272 

test for leucin, 344 
Schick reaction, 605 
Schistocytes, 488 
Schistosomum hematobium in blood, 

585 

in urine, 367 
Schizogone, 564 
Schizont, 572 

Schlosing's method for ammonia, 229 
Schmaltz' specific gravity tubes, 399 
Schmidt and Strasburger's standard 

diet, 100 
Schmidt's fermentation method for 

feces, 124 
Schugner's granules, 569 
Schultze's granular cells, 504, 506 
Sclerostoma duodenale, 151 
Scybala, 105 
Seat worms, 147 
Sebelien's method for protein in milk, 

692 
Secondary anemia, 534 

proteoses, 278 

tubercular pleurisy, 674 
Secretin, 102 
Secretion of gastric juice, 51 

of genital organs, 376 

of mammary glands, 688 

of urine, 176 
Sedimentation, 336 
Sediments in urine, 336 

bacteria, 363 

bilirubin, 345 

blood cells, 354 

calcium carbonate, 349 
oxalate, 341 
phosphate, 346 
sulphate, 345 

casts, 356 

cholesterin, 346 

cystin, 342 

epithelial cells, 349 

fat, 346 

hematoidin, 345 

hippuric acid, 345 

indigo, 257 

leucin, 343 

magnesium ammonium phosphate, 

348 
phosphate, 348 
mucoid material, 349 
mucous threads, 349 
nubecular threads, 180, 349 



Sediments, organized, 349 

parasites, 366 

phosphates, 346, 347 

preservation of, 176 

pus cells, 351 

spermatozoa, 363 

tissue fragments, 363 

tyrosin, 344 

unorganized, 338 

urates, 339 

uric acid, 338 

xanthin, 341 
Sedimentum lateritium, 339 
Seliwanoff's test for levulose, 306 
Semen, 376 

chemistry of, 376 

medico-legal aspects of, 378 

microscopic examination of, 376 

pathology of, 378 

recognition of stains of, 379 

spermatic crystals in, 376 

spermatozoa in, 377 
Seminal stains, 379 

medico-legal aspects of, 378 
Septic pleurisy, 674 
Sero diagnosis, 606 

of pregnancy, 638 

of syphilis, 617 

of typhoid, 607 
Serous cysts of the ovary, 676 

exudates, 664 

plethora, 389 

pleurisy, 664 

sputum, 5 
Serum albumin in blood, 428 
in urine, 261 

diagnosis of syphilis, 617 
of typhoid, 607 

globulin, 274 

determination of, 276 
significance of, 274 
test for, 275 
variations of, 274 

pathology, 593 

reactions, 606 

refraction coefficient of, 389, 429 

special properties of, 593 
Sex, variations of blood cells due to, 

492 
Sexual cycle of malarial parasite, 570 

secretions, 376 
Shadows of leucocytes, 509 

red cells, 486 
Shaffer's method for acetone bodies, 327 
Shiga's bacillus, 132, 713 
Showers of casts, 357 
Side-chain theory of Ehrlich, 594 
Siderosis, 34 

Signet rings in malarial blood, 563, 567 
Significance of acetonuria, 318 

of albuminuria, 261 

of albumosuria, 279 

of Bence Jones proteinuria, 276 

of cylindruria, 357, 362 

of free HC1 in gastric contents, 70 

of globulinuria, 275 

of glycosuria, 284 

of hematuria, 355 

of lactic acid in stomach, 74 



752 



INDEX. 



Significance of leucocytosis, 512 

of levulosuria, 305 

of mucous threads in urine, 349 

of mucus in feces, in 

of nitrogen-partition of urine, 214 

of /?-oxybutyric acid in urine, 325 

of pentosuria, 308 

of pepsin in gastric juice, 78 

of pyuria, 351 
Sjoqvist's method for urea, 225 
Skatoxyl-potassium sulphate in urine, 

258 
Skin, blood in diseases of, 520 

parasites of, 161 
Sleeping sickness, blood in, 575 

organism of, 575 
Small-pox, blood in, 555 
Smears, preparation of, 471 

of blood, 471 

of exudates, 668 

of feces, 119 

of pus, 668 

of sputum, 9 

of syphilitic material, 670 
Smegma bacillus in buccal secretions, 

37 
in exudates, 669 
in sputum, 24 
in urine, 364 
preputii, 669 
Smith's test for bile pigments, 330 
Soaps in feces, 116 
Sodium acid urate, 339 

carbonate as preservative of milk, 

695 

chlorid retention, 193 
in nephritis, 194, 372 
in pneumonia, 194 

in blood, 409 

in urine, 211 
Soft chancre, organism of, 669 
Soluble starch, 36, 82 
Solvents for blood stains, 654 
Specific gravity of blood, 399 

of cerebrospinal fluid, 680 

of exudates, 664 

of milk, 690 

of serum, 399 

of transudates, 663 

of urine, 187 
Specificity of agglutination test, 613 

of complement-fixation test, 633 

of precipitin test, 616 
Spectrophotometer of Hiifner, 416 
Spectroscopic examination, 658 

tests for blood, 410, 658 
Spermatic crystals, 376 
Spermatocele, 678 
Spermatorrhea, 363, 378 
Spermatozoa, 363, 377 
Spermin crystals, 14, 376 
Spiegler's test for albumin, 271 
Spirals of Curschmann, 7, 30, 31 
Spirillum of Asiatic cholera, 61, 129, 

715 
of Obermeier, 574 
of relapsing fever, 574 
of Vincent, 40 
Spirocheta, buccalis, 36 



Spirocheta, pallida, 579 

characteristics of, 579, 582 

cultivation of, 581 

in blood, 579 

in exudates, 669 

in tissues, 669 

staining of, 580, 669 

refringens, 580 
Spit cups, 2 
Spleen, diseases of, blood in, 532 

removal of, 560 
Splenectomy, blood after, 560 
Splenic anemia, 532 
Splenocytes, 503 
Splenomegaly, blood in, 532 

tropical, 576 
Splenomyelogenous leukemia, 539 
Spore cyst, 572 
Spores, staining of, 708 
Sporoblast, 572 
Sporogone, 561 

Sporogony of malarial parasite, 570 
Sporothrix Schenckii, 171 
Sporotrichosis, 171 
Sporozoa in feces, 137 
Sporozoits, 572 

Spotted fever, organism of, 584 
Sputum, 1 

air in, 4 

albumin in, 5 

amount of, 2 
■ bacteria in, 14 

biliary pigments in, 3 

blood in, 3, 5, 11 

character of, 4 

cheesy particles in, 6 

chemistry of, 5 

chromogenic bacteria in, 4 

coal pigment in, 4, 34 

coctum, 32 

collection of, 2 

color of, 3 

concretions in, 8 

consistency of, 2 

cotton fibers in, 4 

crudum, 31 

crystals in, 13 

Curschmann's spirals in, 7, 30, 31 

cytology of, 10 

deportment on standing of, 5 

Dittrich's plugs in, 7 

echinococcus membranes in, 9 

elastic tissue in, 12 

epithelial cells in, 11 

extraneous matter in, 9 

fatty acids in, 13 

ferments in, 6 

ferric oxid in, 4 

fibrinous casts in, 8 

flour in, 4 

foreign bodies in, 9 

fundum petens, 4 

heart disease, cells in, u, 32 

hemoglobin derivatives in, 11, 13 

in abscess of the lung, 33 

in actinomycosis, 27 

in acute bronchitis, 31 

in bronchial asthma, 33 

in broncho-pneumonia, 31 



INDEX. 



753 



Sputum, in chronic bronchitis, 32 

in croupous pneumonia, 31 

in fibrinous bronchitis, 32 

in gangrene of the lung, 33 

in influenza, 33 

in jaundice, 4 

in perforating empyema, 34 

in pneumonoconioses, 34 

in pulmonary tuberculosis, 30 

in putrid bronchitis, 32 

leucocytes in, 10 

macroscopic examination of, 6 

microscopic examination of, 9 

morning, 1 

mucin in, 5 

mucoid, 5 

mucopurulent, 5 

myelin granules in, 11 

nitrogen of, 2 

nummular, 5, 6 

odor of, 4 

origin of, 1 

parasites in, 28 

prune-juice, 3, 31 

purulent, 5 

pus cells in, 10 

reaction of, 3 

red blood cells in, 11 

sanguinous, 5 

serous, 5 

spit-cups for, 2 

stone dust in, 4 

tenacity of, 3 

types of, 5 
Staining characteristics of tubercle 
bacillus, 20 

methods, principles of, 476 

of bacteria, 668 

of blood smears, 478 

of casts, 357 

of elastic tissue, 12 

properties of cells, 496 

vital, 485 
Stains, blood, 478 

Bunge and Trantenroth's, 24 

Burri's, 670 

dahlia, 507 

Ehrlich's tri-acid, 480 
triple, 480 

eosin-hematoxylin, 479 
methylene blue, 478 

Gabbet's, 21 

Ghoreyeb's, 670 

Giemsa's, 483, 670 

Goldhorn's, 580 

Gram's, 668 

india-ink, 670 

iodine, 507 

Jenner's, 481 

Leiner's, 115 

Loffler's methylene blue, 21 

Mallory's, 173 

May-Griinw aid's, 482 

Mullern's, 478 

Neisser's, 39 

Nocht's, 484 

orcein, 12 

osmic acid, 116 

Pappenheim's blood, 481, 484 

48 



Stains, for tubercle bacillus, 21 

polychrome, 481 

Romanowsky's, 481 

safranin, 668 

scharlach R, 116 

seminal, 379 

sudan III, 116 

thionin, 112, 484 

Tunnicliff's, 670 

Turk's iodine, 507 

Unna-Tanzer's, 12 

Van Gieson's, 1 73 

Weigert's fibrin, 8 

Williams and Lowden's, 172 

Wright's, 482 

Zenoni's, 5 

Ziehl-Neelsen, 20 
Staphylococcus pyogenes in sputum, 

26 
Starch in feces, 124 

detection of, 124 

digestion of, 36 

estimation of, 124 
Steatorrhea, 115, 123 
Stegomyia fasciata, 583 
Stercobilin, 107, 255 
Sterility, 378 
Sterilization, 700 
Stippled cells, 498, 567 
Stock vaccines, 724 
Stomach, absorptive power of, 86 

carcinoma of, 91 

contents, 50 

dilatation of, 84, 85 

diseases of, 88 

ectasia of, 85 

fasting, 60 

function of, 84 

histology of , 50 

inflammation of, 90 

motility of, 84 

tube, 52 

ulcer of, 91 

washing, 93 
Stomatitis, catarrhal, 40 

gonorrheal, 41 

mycotic, 41 

ulcerative, 40 

ulceromembranous, 40 
Stone cutters' phthisis, 4 
Stones, bronchial, 8 

gall, 117 

in bladder, 367 

in kidney, 367 

in lung, 8 

intestinal, 118 

in ureter, 367 

in urine, 367 

nasal, 46 

renal, 367 

ureteral, 367 

vesical, 367 
Stools (see Feces), 99 

acholic, 107, 116 

clay-colored, 107 

curds in, 115 

frequency of, 104 

pea-soup, 130 

rice-water, 106, 129 



754 



INDEX. 



Strasburger's method for bacteria in 

feces, 128 
Strauss' test for lactic acid, 75 
Streptococcic sore throat, 40 
Streptococcus hemolyticus, 718 

mucosus capsulatus, 717 

pleurisy, 675 

pyogenes in sputum, 26 
in blood, 591 

viridans in blood, 592 
Strep tothricosis, 27 
Streptothrix eppingeri, 14 
Striatula, 153 

Strongyloides intestinalis, 147 
Strongylus duodenalis, 151 

gigas, 160 

quadridentatus, 151 

renalis, 160 
Structural albuminuria, 265 
Sudanophiles, 509 
Sugar broth, 702 
Sugar-free broth, 702 
Sulphates of urine, 207 

easily split, 207 

ethereal, 207 

preformed, 207 

total,_ 207 

unoxidized, 207 
Sulph-hemoglobin, 412 
Sulphhemoglobinemia, 412 
Sulphocyanates in saliva, 35 
Sulpho-salicylic acid test for albumin, 

27/ 
Sulphur compounds in urine, 206 

amount of, 206 

determination of, 209 

neutral, 208 

types of, 206 

variations of, 207 
Sulphur test for bile in urine, 331 
Surgical interference, blood after, 546 
Syphilis, albuminuria of, 266 

blood in, 558 

hemoglobin test of Justus in, 

559 
organism of, 578 
serum test of Wassermann in, 620 

Tabanus striatus fabricus, 576 
Table for examination of calculi, -?6q 

Gaffky's, 23 
Taeniidae in feces, 140 
taenia aagyptica, 142 

canina, 141 

cucumerina, 141 

cucurbitina, 140 

dentata, 140 

diminuta, 142 

echinococcus, 143 

elliptica, (4.1 

flavopunctata, 142 

inermis, 141 

lata, 144 

leptocephala, 142 

taenia mediocanellata, 141 

minima, 142 

moniliformis, 141 

nana, 142 

saginata, 141 



Taeniidae solium, 140 
varerina, 142 
Tallqvist's hemoglobinometer, 424 
Tape-worms in feces, 139 
Tartar of the teeth, 38 
Taurocholic acid, 117, 191, 331 
Taylor's ash-free diet, 195 
Teichmann's crystals, 657 

test for blood, 657 
Tenacity of sputum, 3 
Tertian malarial organism, 563 
asexual cycle of, 564 
sexual cycle of, 563, 570 
Test meal of Boas, 55 
of Ewald, 54 
of Fischer, 55 
of Riegel, 55 
of Sahli, 56 
of Salzer, 56 
oil, 102 
Testicular casts, 378 
Thecosoma hematobium, 585 
Theobromin, 240 
Theophyllin, 240 
Therapeutic measures, effect of, on 

blood, 494, 516 
Thermolabile substances, 596 
Thermostable substances, 596 
Thionin stain, 112, 484 
Thiosulphuric acid in urine, 191 
Third corpuscles of blood, 523 
Thomas and Weber's method for 

pepsin, 80 
Thoma-Zeiss hemocytometer, 456 
Thorn-apple crystals, 347 
Threads, mucus in urine, 180, 349, 353 
Thread worm, 147 
Throat cultures, 39 
Thrombase, 402 
Thrush, 41 
Tick fever, 584 
Tide, alkaline, of urine, 186 
Timothy bacillus in sputum, 24 
Tinea barbae, 166 
circinata, 166 
favosa, 165 
sycosis, 166 
tonsurans, 167 
versicolor, 169 
Tissue, elastic in sputum, 12 
fragments in feces, 118 
in gastric contents, 63 
in sputum, 9 
in urine, 363 
Toisson's fluid, 460 
Tollen's orcin test for pentose, 310 

naphthoresorcin test for glycu- 

ronic acid, 316 
phloroglucin test, 309 
test for pentose, 309 
Tongue, coating of, 38 
Tonsillitis, leucocytosis in, 515 
Topfer's method for combined HC1, 72 
for free HC1, 68 
test for free HC1, 66 
Total acidity of gastric juice, 64 
components of, 64 
determination of, 64 
limits of, 65 



INDEX. 



755 



Total acidity of urine, 185 
nitrogen of blood, 430 
of feces, 122 
of gastric juice, 82 
in carcinoma, 93 
of urine, 213 
amount of, 213 
determination of, 217 
variations of, 214 
solids of blood, 409 
of feces, 121 
of milk, 691 
of urine, 189, 192 
non-protein nitrogen of blood, 430 
volume of blood, 387 
Towel test for hemoglobin, 416 
Toxemia, hepatic, 222, 286, 305 
intestinal, 123 
renal, 370 
Toxogenic protein decomposition, 216 
Toxoids, 595 
Toxones, 595 
Toxophore, 595 
Trachoma bodies, 48 
Trachyochromatic erythroblasts, 490 
Transfusion, tests before, 653 
Transitional leucocytes, 504 
Transudates, 662 

coagulation of, 664 
obtaining of, 663 
properties of, 663 
Traumatic albuminuria, 265 
Treatment, effect of on Wassermann's 

test, 635 
Trematodes, 156 
in feces, 145 
in sputum, 29 
Treponema pallidum (see Spirocheta 

pallida), 579, 581, 669 
Triacid stain of Ehrlich, 480 

of Pappenheim, 481 
Trichina spiralis, 149 
Trichinella spiralis, 149 
Trichinosis, 149, 520 
Trichiuris trichiura, 149 
Trichocephalus, dispar, 149 
hominis, 149 
mastigodes, 149 
trichiuris, 149 
Trichomonas hominis, 137 
intestinalis, 137 
vaginalis, 381 
in urine, 366 
Trichophyton megalosporon endothrix, 
166 
microsporon, 167 
Trichotrachelidae, 149 
Triple phosphates as calculi, 369 
in sputum, 13 
in urine, 348 
Tripperfaden in urine, 353, 669 
Trombidiosis, 163 
Trombidium irritans, 162 
Trommer's test for glucose, 288 
Tropeolin test for HC1, 67 
Tropical splenomegaly, 576 
Tropics, anemia of, 493 
Trousseau's test for bile pigments, 330 
True albuminuria, 261 



Trypanosoma Brucei, 576 
equiperdum, 576 
Evansi, 576 
Gambiense, 575 
in the blood, 575 
in spinal fluid, 576, 683 
Trypanosomiasis, 575 
Trypsin in feces, 102 

in pancreatic cysts, 678 
in urine, 251 
Tryptophan test, 93 
Tsetse flies, 575 

Tsuchiya's method for albumin, 273 
Tube casts in urine, 356 
Tubercle bacilli in the blood, 592 
in exudates, 667 
inoscopy, 667 
in feces, 132 
in sputum, 17 
culture medium for, 19 
morphology of, 21 
number of, 23 
staining of, 20 

value of examinations for, 22 
in urine, 364 
Tubercular meningitis, 684 

pleurisy, 674 
Tuberculin, 18 

reactions, 601 
Tuberculosis, blood in, 557 
of bladder, 364 
of intestine, 132 
of kidneys, 352 
of lymph glands, 545 
of meninges, 684 
of peritoneum, 676 
of pleura, 674 
pulmonary, blood in, 557 
sputum in, 30 
Tuberculous cystitis, 364 
Tubular insufficiency, 370 
Tumor shreds in feces, 118 
in gastric contents, 63 
in urine, 363 
Tunnel workers' anemia, 153 
Tunni cliff's bacillus, 45 

stain for spirochete, 670 
Turk's iodin stain, 507 

counting chamber, 458 
Two-glass test, 353 
Typhoid bacillus in the blood, 587 
in feces, 130 

Drigalski and Conradi's media, 

130 
Kendall and Day's media, 

in urine, 366 
fever, blood in, 552, 587 
feces in, 130 
Widal reaction in, 607 
pleurisy, 675 
Typhoidin reaction, 605 
Ty rosin in sputum, 13 
in urine, 246, 344 

Uffelmann's test for lactic acid, 75 
Uhlenhuth's antiformin method for 

tubercle bacilli, 18 
Ulcer of the stomach, 91 



756 



INDEX. 



Ulceromembranous angina of Vincent, 

40 
Unaltered bile in feces, 108 
Uncinaria Americana, 152 

duodenalis, 151 
Uncinariasis, 151 

Undetermined nitrogen of urine, 246 
Unilateral nephritis, 371 
Unit of counting chamber, 463 
Unna-Tanzer's stain, 12 
Unorganized sediments in urine, 338 
Unoxidized sulphur of the urine, 207 
Uranium method for phosphates, 202 
Urates in urine, 339 
Urea in blood, 435 

in urine, 219 
amount of, 220 
determination of, 221 
variations of, 220 
Urease method for urea, 225, 436 
Uremia, blood in, 437 

urine in, 370 
Uremic coefficient, 438 

constant, 438 
Ureometer of Doremus, 222 

of Hinds, 223 
Ureteral calculi, 367 
Ureteritis membranacea, 252 
Urethritis, anterior, 353 

posterior, 353 
Uric acid, 232 

calculi, 368 

diathesis, 234 

in blood, 439 

in the urine, 232 

determination of, 235 
metabolism of, 232 
variations of, 233 

sediment, 338 
Uricacidemia, 442 
Urine, 175 

acetone in, 191, 320 

acidity of, 184 

albumin in, 261 

albumoses in, 276 

alkaline tide of, 186 

alkapton bodies in, 182, 333 

alloxur bodies in, 191, 240, 341 

amino-acids in, 246 

ammonia in, 227 

amount of, 177 

animal gum in, 314 

animal parasites in, 366 

appearance of, 180 

ash of, 192 

bacteria in, 363 

Bence-Jones protein in, 276 

bile acids in, 331 

biliary pigments in, 329 

black, 182 

blood cells in, 354 
pigment in, 328 

blue, 183, 257 

calcium in, 211 

calculi in, 367 

carbohydrates in, 283 

carbonates in, 211, 349 

casts in, 356 

changes on standing of, 180 



Urine, chemistry of, 190 

chlorids in, 192 

cholesterin in, 346 

chromogens in, 254 

chyle in, 182, 346 

clearing of, 298, 300 

collection of, 176 

color of, 181 

composition of, 190 

consistence of, 181 

creatin in, 241, 245 

creatinin in, 241 

cryoscopy of, 371 

cultures of, 363 

cystin in, 208, 342, 370 

dextrin in, 314 

dextrose in, 284 

diacetic acid in, 324 

diastase in, 251 

drug reactions in, 183 

Ehrlich's benzaldehyde reaction 
in, 336 
diazo reaction in, 334 
egg-yellow reaction in, 335 

electric conductivity of, 371 

epithelial cells in, 349 

fat in, 346 

fatty acids in, 249 

ferments in, 251 

fibrin in, 283 

foreign bodies in, 366 

free mineral acidity of, 185 
organic acidity of, 185 

functional diagnosis from, 370 

glucose in, 284 

glycosuric acid in, 333 

glycuronic acid in, 314 

green, 182 

hematoporphyrin in, 328 

hemoglobin in, 288, 328 

hippuric acid in, 248, 345 

homogentisic acid in, 182, 333 

indican in, 256 

indigo in, 256 

inosite in, 314 

iron in, 213 

lactic acid in, 249 

lactose in, 312 

laiose in, 306 

leucin in, 246, 343 

leucocytes in, 351 

levulose in, 305 

magnesium in, 211 

maltose in, 313 

melanin in, 182, 332 

microscopy of, 336 

mucin-like substances in, 252 

mucoid material in, 349 

neutral sulphur in, 208 

nitrogen in, 213 

nitrogenous bodies in, 213 

nubecula in, 180, 349 

nuclein bodies in, 240 

nucleo-albumin in, 252 

odor of, 183 

optical activity of, 190 

organized sediments of, 349 

oxalic acid in, 250 

oxaluric acid in, 250 



INDEX. 



757 



Urine, /?-oxybutyric acid in, 325 

parasites in, 366 

pentoses in, 308 

peptone in, 281 

phosphates in, 198, 346, 347 

physical properties of, 177 

pigments in, 254, 328 

potassium in, 211 

preservation of, 176 

protein of, 260 

proteoses in, 276 

ptomaines in, 342 

purin bases in, 240 

pus in, 351 

quantity of, 177 

reaction of, 184 

Russo's reaction in, 335 

sediments of, 336 

serum-albumin in, 261 
globulin in, 274 

skatoxyl in, 258 

sodium in, 211 

solids of, 189, 192 

specific gravity of, 187 

spermatozoa in, 363 

sugar in, 284 

sulphates in, 207 

sulphur compounds in, 206 

tissue fragments in, 363 

total solids of, 189, 192 

tyrosin in, 246, 344 

urates in, 338 

urea in, 219 

uric acid in, 232, 338 

urobilin in, 255 

urochrome in, 254 

uroerythrin in, 254 

urohematin in, 256 

urorosein in, 260 

xanthin bases in, 240, 341 
Urinod, 183 
Urinometer, 188 
Urinous odor, 183 
Urobilin, 255 
Urobilinuria, 255 
Urochrome, 254 

Urochromogen, Weisz' test for, 254 
Uroerythrin, 254 
Uroferric acid, 208 
Uroleucic acid, 191, 333 
Urophain, 260 
Urorhodin, 258 
Uroroseinogen, 260 
Urorubin, 258 
Urostealith calculi, 370 
Uterine secretions, 382 

Vaccination, antityphoid, 600, 726 
Vaccines, 600, 723 

autogenous, 725 

diagnostic use of, 600 

preparation of, 724 

stock, 724 
Vaccine therapy, 600 
Vacuolization, 497 
Vaginal secretions, 380 
Vaginitis, catarrhal, 380 

gonorrheal, 381 
Value of blood examinations, 659 



Value of functional renal diagnosis, 370 

of search for tubercle bacilli, 22 
Van Deen's test for blood, 109, 655 
Van Ermengem's stain for flagella, 708 
Van Gieson's stain for Negri bodies, 173 
Van Slyke and Cullen's method for urea, 

226 
Vaquez' disease, 496 
Variations in number of leucocytes, 

5ii, 521 

of red cells, 492, 500 
Variola, blood in, 555 
Venous blood, 394 

puncture, 393, 585 
Vermiculus, 570 
Vernal conjunctivitis, 49 
Vesicular albuminuria, 261 

mole, 384 
Vincent's angina, 40 

bacillus, 40 

spirillum, 40 
Vinegar eel in urine, 366 
Viscosity of blood, 401 
Vitali's test for pus, 353 
Vital staining of blood cells, 485 
Volatile alkalinity of urine, 187 
Volhard's method for chlorids, 195 
Volume index of blood, 391 

of blood, 387 

quotient, 391 

value, 391 
Vomitus, 60 

bile in, 61 

blood in, 61 

fecal, 61 

green, 61 

mucus in, 61 

odor of, 61 

pancreatic fluid in, 61 

parasites in, 61 

pus in, 61 

rice water, 61 

Wagner's test for blood, in 
Wang's method for indican, 259 
Warn eld's test for pregnancy, 247 
Wassermann's serum reaction for 

syphilis, 620 
Waxy casts, 358 
Weber's test for blood, no 
Weidel's test for xanthin, 341 
Weinstein's test for gastric carcinoma, 

93 
Weisz' test for urochromogen, 254 
Welch's gas bacillus, 133 
Weyl's test for creatinin, 243 
Whetstone crystals of uric acid, 339 

of xanthin, 341 
Whip worm, 149 
White blood cells (see Leucocytes) , 

502 
Whooping cough, bacillus of, 26, 711 

blood in, 556 
Widal reaction, 607 
Williams and Lowden's stain for Negri 

bodies, 172 
Williamson's blood test in diabetes, 548 
Winternitz method for gastric motil- 
ity, 86 



758 



INDEX. 



Winternitz', Henry and McPhedrans' 

test for catalase, 522 
Wolff and Junghans' test, 95 
Wright's opsonic method, 599 
stain for blood smears, 482 
vaccine therapy, 600 
Wright and Kinnicutt's method for 
blood plates, 469 

Xanthin bases in feces, 123 
in urine, 191, 240, 341 
calculi, 370 
Xerosis bacillus, 40 
Xylose in urine, 308 



Yaoita's method for ova, 133 
Yeast cells in feces, 126 

in gastric contents, 62, 63 

in sputum, 15 

in urine, 293 
Yellow fever, blood in, 583 

mosquito theory of, 583 



Ziehl-Neelsen method for tubercle 

bacilli, 20 
Zygotes, 572 

Zymogens in gastric juice, 77, 81 
Zymophore, 598 



